Article

Purification of Rat Liver Acetyl Coenzyme A Carboxylase and Immunochemical Studies on its Synthesis and Degradation

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Abstract

Acetyl coenzyme A carboxylase from rat liver was isolated in homogeneous form as evidenced by sedimentation velocity experiments and Ouchterlony double diffusion analyses. The specific activity and biotin content of the purified enzyme preparation were similar to those of pure chicken liver enzyme isolated previously. Antibody against rat liver acetyl-CoA carboxylase was prepared by injecting rabbits with the purified enzyme. With the use of this antibody, the mechanisms were studied by which the level of liver acetyl-CoA carboxylase activity was varied in fasted rats, in those subsequently refed a fat-free diet and in diabetic rats. Immunochemical analysis demonstrated that the wide variations in the level of acetyl-CoA carboxylase activity in liver extracts derived from rats under the different dietary and hormonal conditions were accompanied by proportionate changes in the quantity of immunochemically reactive protein. This indicates that the changes in the activity level are determined by changing quantities of the carboxylase protein. Isotopic leucine incorporation studies showed that the relative rate of synthesis of liver acetyl-CoA carboxylase was decreased 1.9-fold and 1.7-fold by fasting and diabetes, respectively, whereas increased 4.0-fold by fat-free refeeding. The rate of degradation of the carboxylase, expressed as half-life, was found to be essentially the same in normal, refed and diabetic rats, i.e. 59, 55 and 59 h respectively, and accelerated in fasted rats, i.e. 31 h. Thus, the increase or decrease in the carboxylase quantity in refed or diabetic animals, which are presumably in a steady state, can be ascribed to a rise or fall in the rate of enzyme synthesis, whereas the decrease in the carboxylase quantity in fasted animals, which are not in a steady state, is due both to diminished enzyme synthesis and to accelerated enzyme degradation. On the basis of these results, it is discussed that the control of enzyme quantity by changes in the rate of enzyme degradation may play an important role when the animal deviates from a steady state for adjustment to a new environment.

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... The half-life o f ACC varies from 1 -3 days according to metabolic status (Nakanishi and Numa, 1970 (Munday and Hardie, 1986a;Munday and Williamson, 1982;McNellie and Zammit, 1982). Liver total ACC activity decreased after 48 hours starvation and rose upon 24 hours refeeding (Munday et cd, 1991). ...
... This restoration o f total activity could be prevented by treatment with puromycin, a protein synthesis inhibitor or actinomycin D, a transcription inhibitor (Hicks et d , 1965), suggesting participation o f protein synthesis in this regulation. Starvation o f rats for 48 hours followed by feeding with a fat-free diet, induces the synthesis and activity o f liver ACC, due to simultaneous changes in the rate o f degradation and synthesis o f ACC (Nakanishi and Numa, 1970). Diet induced changes in the concentration o f ACC in liver or epididymal fat pads correlated positively with the amount o f ACC mRNA (Pape et d, 1988). ...
... The (Gibbons and Pullinger, 1986;Clarke et al, 1977;Willumsen et d , 1993;Roach et d, 1987 (Zakim et d , 1970) Comparable inhibition o f ACC in cultured hepatocytes incubated for 60 min with gemfibrozil indicated that the effect was due directly to gemfibrozil or its metabolite and was not the result o f hormonal changes in vivo. Such a rapid effect implicates an allosteric or covalent mechanism o f inhibition, rather than a change in enzyme concentration since ACC has a relatively long half-life (1-3 days) (Nakanishi and Numa, 1970). It was possible to directly inhibit ACC with gemfibrozil in its assay. ...
Thesis
The liver plays a central role in the production and metabolism of lipoproteins, with adipose tissue also contributing a large proportion of whole body fatty acid synthesis, in rat. Cholesterol and triacylglycerol are major lipid components of lipoproteins and inhibition of their hepatic synthesis is a major effect of many hypolipidaemic drugs in the treatment of atherosclerosis. The molecular mechanisms of inhibition of hepatic fatty acid and cholesterol biosynthesis by gemfibrozil and related hypolipidaemic agents and effects on general carbohydrate and lipid metabolism were investigated. Administration of the drug gemfibrozil (ip) produced a rapid (90 min) inhibition of fatty acid synthesis (68%) and cholesterol synthesis (81%) in rat liver in vivo. In primary rat hepatocyte cultures gemfibrozil and clofibrate were potent inhibitors of fatty acid synthesis, exhibiting maximal inhibition of 82% and 50%, respectively, with IC50 values of 0.6 mM and 1 mM, respectively. Similarly, gemfibrozil and clofibrate produced maximal inhibition of cholesterol synthesis of 90% and 68%, with IC50 values of 0.25 mM and 0.5 mM, respectively. Gemfibrozil and clofibrate have structures analogous to those of fatty acids. Another potent hypolipidaemic agent is MaxEPA which is composed largely of the fish oil n-3 eicosapentanoic acid (EPA). EPA and its parent compound linolenic acid produced 92% and 66% inhibitions of fatty acid synthesis in hepatocytes. Gemfibrozil stimulated the phosphorylation and inactivation of hepatic acetyl- CoA carboxylase (ACC), the regulatory enzyme of fatty acid synthesis, both in vivo and in hepatocytes. In vivo, gemfibrozil produced a 60% inhibition of HMG-CoA reductase activity in microsomes reducing 'total' activity by 30% and 'expressed' activity by 57%. This suggested the combination of phosphorylation and a decrease in enzyme concentration as the mechanism of inhibition. AMP-activated protein kinase (AMP-PK) phosphoiylates and inactivates both of these regulatory enzymes physiologically in liver. Gemfibrozil was observed to activate AMP-PK by 100 - 200% both in liver in vivo and in hepatocytes. Experiments with partially purified AMP-PK suggest that this activation is due to increased AMP-PK phosphorylation. Gemfibrozil clearly activates the AMP-PK cascade in rat liver. Gemfibrozil is known to stimulate fatty acid oxidation. This was confirmed by a 46% increase in circulating ketone bodies in gemfibrozil-treated rats in vivo and a 40% increase in ketone body production in hepatocytes treated with either gemfibrozil (1 mM) or clofibrate (5 mM). An additional mechanism in this partitioning of fatty acids away from esterification was demonstrated in vivo by a 58%> inhibition of hepatic glycerol-3-phosphate acyltransferase (GPAT). Phosphorylation and inactivation of GPAT by AMP-PK could not be demonstrated. Gemfibrozil treatment of rats for 150 min brought about a 15% rise in plasma glucose, a 14% rise in hepatic glycogen content and a 68% increase in the rate of hepatic glycogen synthesis. This indicated that decreased carbohydrate utilisation by liver in response to gemfibrozil requires compensatory measures by other pathways (and perhaps other tissues for example, white adipose tissue) and may have serious implications for gemfibrozil treatment of diabetic hyperlipidaemia.
... Long term control o f ACC is achieved via changes in the mRNA levels and in the rate o f protein synthesis and degradation (Majerus and Kilbum, 1969). The half-life o f ACC was found to vary fi'om 1-3 days according to metabolic status (Nakanishi and Numa, 1970). The rise in ACC concentration in liver upon refeeding after starvation can be prevented by treatment with puromycin, a protein synthesis inhibitor or actinomycin D, a transcription inhibitor (Hicks et al, 1965). ...
... The rise in ACC concentration in liver upon refeeding after starvation can be prevented by treatment with puromycin, a protein synthesis inhibitor or actinomycin D, a transcription inhibitor (Hicks et al, 1965). A fat-free diet induces synthesis o f ACC and increases its activity in rat liver o f 48 hour-starved animals due to simultaneous changes in the rate o f degradation and synthesis o f ACC (Nakanishi and Numa, 1970). Diet induced changes in the concentration o f ACC in rat liver or epididymal fat pads also correlate positively with changes in the amount o f ACC mRNA (Pape et al, 1988). ...
... Starvation is associated with inhibition of fatty acid synthesis in rat liver and the effect is reversed by refeeding for 2-4 hours (Holness et al, 1988;Holness and Sugden, 1990). While starvation is accompanied by decreased hepatic ACC concentration (Nakanishi and Numa, 1970;Munday et al, 1991), the fed to starved and starved to refed transitions are paralleled by phosphorylation and inactivation, and dephosphorylation and activation of hepatic ACC, respectively (Jamil and Madsen, 1987;Thampy and Wakil, 1988;Munday et al, 1991). ...
Thesis
The lactating rat mammary gland is highly active in synthesising fatty acids and isolated mammary acini were used to investigate the regulation and interdependence of specific regulatory steps in the pathway from glucose to fatty acids. Various putative effectors of lipogenesis were used to investigate the control and relative importance of these specific regulatory steps. Acetoacetate, a potential physiological signal in starvation inhibited fatty acid synthesis (38%) but not acetyl-CoA carboxylase (ACC). Surprisingly, ACC was activated and this survived purification (Vmax increased by 100%). ATP-citrate lyase (ATP-CL) and pyruvate dehydrogenase (PDH) were inhibited by 18% and 83%, respectively. However, neither of these are likely to account for the lipogenic inhibition, given the increase in citrate concentration caused by acetoacetate. Glucose uptake was reduced by 54% in the presence of acetoacetate, but this was not the result of a decrease in the rate of glucose transport which was unaffected. The data suggest that reduced glucose supply to the cells is the major factor in lipogenic inhibition, possibly via the consequent reduced production of NADPH for fatty acid synthesis, or glycerol-3-phosphate for esterification. Inhibition of glycolysis probably occurs as a result of phosphofructo-1-kinase (PFK-1) inhibition by the elevated citrate levels in combination with PDH inactivation. Investigation of the profile of protein phosphatase activity in mammary gland showed the presence of okadaic acid-sensitive PP1 and PP2A. Okadaic acid is a potent cell-permeable inhibitor of PP1 and PP2A. It inhibited fatty acid synthesis (63%) without affecting the rate of glucose uptake by rat mammary acini. While GLUT 1 (the predominant glucose transporter in mammary acini) like GLUT 4 (the insulin stimulated glucose transporter) can be regulated by translocation to the plasma membrane, these studies confirm that phosphorylation is unlikely to be involved in this regulation. ACC was phosphorylated and inactivated (Vmax decreased by 70%) in response to okadaic acid. This is an expected result of PP2A inhibition but a transient activation of AMP-PK was also observed. Time course studies showed that PDH inhibition in response to okadaic acid was a consequence of the inhibition of ACC, presumably via an increased acetyl CoA/CoA ratio that would activate PDH kinase. The polyunsaturated fatty acid, eicosapentanoic acid (EPA), inhibited fatty acid synthesis in mammary acini in a dose-dependent manner. Maximal inhibition was achieved at 1mM EPA and at this concentration both PDH and ACC were inhibited. Linolenic and linoleic acids produced similar inhibitory effects with the degree of inhibition depending on the degree of unsaturation of the fatty acid. Clofibrate and gemfibrozil are lipid-lowering drugs with structures analogous to those of fatty acids. These drugs also inhibited fatty acid synthesis in acini (33% and 84%, respectively) via PDH and ACC inactivation. In vivo studies with gemfibrozil indicated that doses of this agent known to produce peroxisomal proliferation produced similar short term inhibition of fatty acid synthesis (68%), and cholesterol synthesis (81%) in rat liver in vivo. Surprisingly, fatty acid synthesis in white adipose tissue was increased by 71%.
... Purification of ACC-1: Performed through protein precipitation by 40% Ammonium sulfate, was dissolved in phosphate buffer at (0.1 M,PH 7.8) and proceeded into a further purification by applying into a Sephadex G-100 Column (2.5 x 40 cm) using phosphate buffer ( PH 7.8, 0.1 M) as elution buffer. The resultant was extra purified by Ion Exchange Chromatography using DEAE-Cellulose (2.5 x 25 cm) and a washing solution consisting of phosphate buffer Solution (20mM, PH7.8) which contains (10 mM of sodium citrate) and the dilution solution that consists of phosphate buffer (0.13M,0.75M ,pH7.8) which contains (10mM) of sodium citrate (12,13,14) Electrophoresis: Used Garfine method for electrophoresis Polyacrylamide under non denatured condition (15) The molecular weight of the purified enzyme ACC-1 was measured compared to standard proteins standard proteins solution: Prepare to dissolve standard proteins Bovine albumin, albumin Eggs, Chymotrypsinogen, Lysozyme, Glucose Oxidas, Alcohol dehydrogenase and Immunoglobulins In the sample solution at a concentration of 5 mg / ml.. Temperature Effect: The effect of temperature on the purified enzyme(ACC) from sera at different temperatures (20,25,30,37,40,45,50•C) and the temperature was adjusted by thermostat and the activity was measured used Kroeger method (11) . The activation energy (Ea) is calculated by drawing the relationship between 1 / T (K-1) against log Vmax as shown in Figure ( pH Effect : The studied effect of the acid function on the purified serum solution the reaction mixture solutions were prepared in different acid function (4.5 ,5,5.5,6,6.5,7,7.5, 8,8.5,9) and the pH of the prepared solutions was adjusted by Hydrochloric acid 1M and sodium hydroxide 1M and the activity was measured used Kroeger method at optimum temperature. ...
... The result of ACC-1purification protocol could be illustrated in table1 , Fig1and Figure 1: Gel filtration of ACC1 from breast cancer serums blood in premenopausal group before . Mastectomy or treatment 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 These results are agree with the results of Roessler, whe obtained one single peak of the purified ACC enzyme from the filtered Cyclotella Cryptica by gel filtering method (14) . It also agreed with Dehaye and his group where he obtained one single peak of the purified ACC enzyme from the seeds of the gel filtration method (16). ...
Article
Full-text available
Acetyle CoA Carboxylase-1 was purified from sera of premenopausal women with breast cancer( before Mastectomy or treatment ) by Gel Filtration using Sephadex G-100 and by Ion Exchange using DEAE-Cellulose A-50, also the molecular weight was estimated by the Acrylamide Electrophoresis in the absence of denaturing elements . The result showed that a single band was obtained at 220KD by Gel Filtering while Ion Exchange showed one band at 200KD. The optimum temperature of purified Acetyle CoA Carboxylase-1 was 40 °C, optimal pH at 7.5 and the optimum substrate concentration at 1.8mM. Michaelis-Menten constant (km) was 0.37Mm and Velocity Maximum (Vmax) was 25mM.min ⁻¹ The Activation energy(Ea) was 28 KJ/mol.
... Again, these results demonstrate a difference in the mechanism of action of the malonyl-CoA produced from the two ACC isoforms, although malonyl-CoA from ACC-2 can clearly be used for fatty acid synthesis in the absence of ACC-1. [77,78]. Using isotopic leucine incorporation and immunochemical titration experiments, they showed that changes in total ACC activity correlated well with immunodetectable protein, and they inferred there were no changes in ACC catalytic efficiency. ...
... The model developed as a result of these studies, provides a mechanism to explain the degradation of ACC during nutrient starvation and therefore to the increase in turnover of ACC during catabolic stress. Even so, ACC turnover is known to be slow, with the half time of approximately 24 hours or greater even during starvation or insulin deficiency [77,78]. This mechanism is unlikely, therefore, to contribute to short-term ACC control. ...
... Nous avons pu constater en effet qu'une simple préincubation avec le citrate suffit à lever l'inhibition exercée par le jeûne sur l'acétyl CoA carboxylase du tissu adipeux de rat, alors qu'elle était sans effet sur le foie, ce qui laisserait supposer que la concentration d'ACX est moins vite abaissée par le jeûne dans le tissu adipeux que dans le foie. Ce serait en général (diabète, obésité), plus la synthèse que la dégradation qui conditionnerait l'activité de l'enzyme (Nakanishi et Numa, 1970 ;Craig et al., 1972). Les dégradations de ces enzymes ne seraient efficaces comme objet de régulation qu'à jeun (Majerus et Kilburn, 1969). ...
... Les quelques articles consacrés à l'étude de l'action des hormones sur la synthèse des enzymes dans le tissu adipeux de rat ont montré que, contrairement à ce qui se passait dans le foie, l'activité de la FAS et celle de l'ACX du tissu adipeux de rat étaient abaissées par administration de glucocorticoïdes (Diamant et Shafrir, 1975 ;Volpe et Marasa, 1975), alors que dans le foie c'est le glucagon qui exerce une action inhibitrice sur la synthèse de ces enzymes (Volpe et Marasa, 1975 ;Lakshmanan et al, 1972). En revanche l'insuline stimule la synthèse de la FAS et de l'ACX aussi bien dans le foie que dans le tissu adipeux (Nakanishi et Numa, 1970 ;Lakshmanan et al., 1975); des effets divergents ont été obtenus suivant les auteurs en ce qui concerne l'action des hormones thyroïdiennes (Roncari et Murthy, 1975 ;Diamant et al., 1972). ...
... ACC has been purified and characterised from a variety of mammalian tissues (Nakanishi & Numa 1970, Inoue & Lowenstein, 1972, Hardie & Cohen, 1978Bianchi et al, 1990 and two immunologically distinct isozymes of ACC have been identified (Thampy, 1989 andBianchi g/ a/, 1990). ...
... Long term control of ACC 265 in liver involves changes in protein synthesis and degradation (Nakanishi & Numa, 1970). Short term regulation includes allosteric regulation (by citrate and long chain fatty acyl-CoA) and covalent modification (phosphorylation/dephosphorylation) (Allred & Riley, 1997) ...
Thesis
Three skeletal muscle fibre types (type I, type IIa and type IIb) were assayed for the presence of the following enzymes ATP-citrate lyase, citrate-dependent acetyl-CoA carboxylase, fatty acid synthase and malonyl-CoA decarboxylase. All activities were present in each of the muscle fibre types studied. A cytosolic activity of malonyl-CoA decarboxylase would appear to offer a feasible route for the disposal of malonyl-CoA in skeletal muscle. Rat soleus muscle strips were incubated with 5mM glucose followed by measurements of tissue contents of malonyl-CoA, long chain fatty acyl-CoA and carnitine esters. Alternatively muscle strips were incubated with 5mM glucose and 0.2mM palmitate followed by measurements of [14C] palmitate CO2 formation from exogenous palmitate or from fatty acids released from prelabelled glycerolipids. Etomoxir at high concentrations (150µM) significantly decreased the malonyl-CoA content by 50% and at low concentrations (50 & 100µM) had no effect on malonyl-CoA. Etomoxir had no effect on the total long chain ester pool but significantly increased long chain acyl-CoA and decreased the ratio of acyl-carnitine/acyl-CoA suggesting that such changes could be diagnostic for inhibition of CPT 1. Insulin and DC A (3mM) increased both malonyl-CoA and long chain fatty acyl-CoA content and decreased the ratio of fatty acylcarnitine/acyl-CoA and β-oxidation. Isoprenaline and palmitate (0.5mM) opposed the effects of insulin, by decreasing the contents of malonyl-CoA and long chain fatty acyl-CoA, increasing the ratio of fatty acylcarnitine/ acyl-CoA and increasing β-oxidation. These findings are consistent with the notion that all these agents can cause acute regulation of CPT 1 in type I skeletal muscle. In the presence of 5-amino-4-imidazolecarboxamide ribonucloeside (AICAR) (1mM) the malonyl-CoA content decreased by 65% and decreased the content of both long chain esters significantly, in particular long chain acyl-CoA. Oxidation of 14C-labelled exogenous and endogenous fatty acid was measured in soleus muscle strips incubated with insulin as mentioned above. Isoprenaline (10-7M) increased both processes (28% and 103% respectively). Indicating that isoprenaline has a lipolytic effect. AICAR (1mM) increased oxidation of exogenous fatty acid by 102% but had no effect on endogenous oxidation. It is therefore concluded that AICAR causes a decrease in lipolysis in muscle. Similar experiments led to the suggestion that dichloroacetate (3mM) had a lipolytic effect in muscle.
... The rat-liver enzyme was studied by Lynen's group (Numa et al., 1964;Matsuhashi et al., 1964) and isolated in homogeneous form by Nakanishi and Numa (1970) and by Inoue and Lowerstein (1972). The enzyme possesses two catalytic sites: one at which the biotin prosthetic group is carboxylated at the 1'-N-atom (Numa et al., 1964) and one at which the carboxyl group is transferred to acetyl-CoA to yield malonyl-CoA. ...
... The former effect was explained by Bortz and Lynen (1963b) on the basis of an increased level oflong-chain acyl-CoA in fasted liver. However, Nakanishi and Numa (1970) demonstrated that wide variations in the activity of acetyl-CoA carboxylase in liver extracts from rats under different dietary and hormonal conditions were accompanied by proportionate changes in the quantity of protein that was immunochemically reactive with an antibody against rat liver acetyl-CoA carboxylase. This indicates that these changes in activity are determined by changing quantities of the carboxylase protein rather than by short-term regulatory effectors like acyl-CoA. ...
Article
The liver occupies a central position in the metabolism of lipids in mammals. Besides its ability, like most tissues, to utilize fatty acids obtained from the blood as an energy source by oxidizing them completely to carbon dioxide and water, the liver has the unique capacity to convert these fatty acids into ketone bodies. It thus supplies the extrahepatic tissues with a major energy source, especially under conditions of limited availability of glucose.
... Starvation is a physiological situation which results in the inhibition of lipogenesis in rat liver and this inhibition is reversed upon chow refeeding for 2-4 hours (Holness et al. 1988, Holness and. It has been shown in the livers of starved rats that the concentration of ACC is decreased and this is probably due to both decreased synthesis and increased degradation (Nakanishi and Numa, 1970) but it has also been shown that the phosphorylation state of hepatic ACC varies with nutritional condition. Hepatic ACC activity decreased upon starvation and there was a concomitant increase in phosphate content with a value of 7.6 molP/ mol subunit obtained for 48-hour starved rats. ...
... This is in agreement with previous reports Hardie, 1986, Williamson et al. 1983). The delayed onset o f decreased ACC concentration might be predicted for an enzyme with a halflife in excess of 24 hours (Nakanishi and Numa, 1970) however the equally slow inactivation o f this enzyme compared with PDH is surprising. W ork conducted on the purifed enzyme confirms that there is no inactivation of ACC during the first 6hours o f starvation (Fig. 4.4). ...
Thesis
Lipid biosynthesis in the mammal is stringently regulated according to nutritional status. The rate limiting step in fatty acid synthesis is believed to be that catalysed by acetyl-CoA carboxylase (ACC). The activity of ACC is regulated both allosterically and by reversible phosphorylation. The work described in this thesis was conducted to ascertain which protein kinases are responsible for the in vivo phosphorylation and inactivation of ACC in the major lipogenic tissues i.e. lactating mammary gland and liver. Glucagon-mediated inhibition of ACC in hepatocytes, and phosphorylation and inactivation of purified ACC in vitro by purified cAMP-dependent protein kinase (cAMP-PK) from bovine heart suggested cAMP-PK as a good candidate for the physiological ACC kinase. However, intensive study of the catalytic subunit of cAMP-PK from mammary gland showed that it is probably an isozymic form of cAMP-PK characterised by poor stability and different substrate specificity to its cardiac counterpart, in particular a low affinity for ACC. Furthermore it appears that there is present in lactating mammary gland a factor that specifically inactivates the free catalytic subunit of cAMP-PK which may also be a reason for its lack of effect on ACC in vivo. Under physiological conditions eg over time courses of starvation, the inactivation of ACC in lactating rat mammary gland or rat liver more closely correlated with activation of AMP-activated protein kinase (AMP-PK) than cAMP-PK, This provided further evidence that AMP-PK and not cAMP-PK is the physiological ACC kinase. This work also suggested that the primary control point in the lactating mammary gland is pyruvate dehydrogenase (PDH) and its associated kinase, but in the liver ACC is the primary control point. AMP-PK itself is regulated by reversible phosphorylation, being phosphorylated and activated by a separate kinase kinase activity. A partial purification and characterisation of the AMP-PK kinase from mammary gland is described, together with its stimulation by fatty acyl-CoA. The final products of mammalian lipid biosynthesis are triglycerides and cholesteryl ester, these are assembled together to form very low density lipoprotein (VLDL) in the liver and are then secreted from the liver into the blood stream. The major structural protein of VLDL is apolipoprotein B-100 (apo B). Apo B is known to be a phosphoprotein in vivo. This work describes the in vitro phosphorylation of apo B by cAMP-PK and AMP-PK and identifies the sites of phosphorylation. It also reports the partial characterisation of a possibly novel apo B kinase.
... Acetyl-CoA carboxylase (ACC) activity in hepatic cytosol fractions was determined by the H 14 CO 3 − fixation assay (Nakanishi and Numa, 1970). Fatty acid synthase (FAS) activity in hepatic cytosol fractions was determined by the spectrophotometric method (Nepokroeff et al., 1975). ...
Article
In this study, we investigated the effects of dietary soybean-germ protein (SGP) on abdominal fat accumulation in growing broiler chickens. Atotal of 24 seven day-old male broiler chicks were allocated to two cages and fed a control diet or a SGP diet for 21 days. Dietary SGP significantly decreased the weight of abdominal adipose tissue. Hepatic triglyceride content and plasma levels of triglyceride and very low density lipoprotein-triglyceride were significantly decreased by dietary SGP. The enzymatic activity and mRNAlevel of fatty acid synthase in the liver were significantly decreased by dietary SGP. These results suggest that dietary SGP might reduce abdominal fat accumulation by downregulation of fatty acid synthesis in the liver of growing broiler chickens. The weights of breast muscles and thighs were significantly increased by dietary SGP. The ribosomal capacity of the breast muscles was significantly increased by dietary SGP. These results suggest that dietary SGP might increase skeletal muscle weight by upregulation of protein synthesis in the muscle of growing broiler chicken. All our findings suggest that SGP can be used as a new feed ingredient for broiler chickens.
... Biotin is covalently attached to this family of enzymes by biotin protein ligase (BPL). Biotin-dependent carboxylases have half-lives of 1-8 days [Majerus and Kilburn 1969;Nakanishi and Numa 1970;Weinberg and Utter 1979;Weinberg and Utter 1980;Freytag and Utter 1983]. Both ACC1 and ACC2 catalyze the incorporation of bicarbonate into malonyl-CoA, a key regulatory step in fatty acid synthesis (ACC1) and mitochondrial fatty acid transport (ACC2). ...
Article
Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to carboxylases and histones in eukaryotic cells. Biotinylated carboxylases play essential roles in the metabolism of fatty acids, amino acids, and glucose; biotinylated histones play essential roles in gene regulation and genome stability. HCS null individuals are not viable whereas HCS deficiency is linked to developmental delays and phenotypes such as short life span and low stress resistance. Greater than 2,500 single nucleotide polymorphisms (SNPs) have been reported for HCS, but the biological importance of these polymorphisms is unknown. We hypothesized that some of these SNPs impair catalytic activity and that this effect can be overcome by dietary intervention with biotin. Here, we analyzed the enzyme kinetics of five recombinant HCS variants using a propionyl-CoA carboxylase surrogate (“p67”) as substrate for biotinylation. Vmax of variants L216R, V96F and G510R were 6%, 78% and 73%, respectively, of the Vmax in wild-type HCS. The Km values of the variants V96F and G510R were not significantly different from wild-type HCS. The activity of L216R was too low to allow for meaningful analysis of Km. In contrast, the affinity of variant Q699R for biotin was significantly lower than that of wild type HCS (Km: 1.57 times that of wild type) and its Vmax could be restored to that of wild-type HCS by biotin supplementation. This is the first biochemical characterization of catalytic activities of HCS variants. Also, this is the first report to show that HCS activity can be restored to normal by biotin supplementation. Advisor: Janos Zempleni
... The oncogene-mediated activation of SREBP was reflected in elevated transcript (Figures 2b and S1a) and protein ( Figure 2a) levels of three canonical SREBP targets, ACC1, FASN and SCD. While the transcript levels of ACC1, FASN and SCD were all sensitive to mTORC1 inhibitors, only SCD was decreased at the protein level following overnight treatment, reflecting the longlived nature of the ACC1 and FASN proteins 35,36 . ...
Article
An enhanced capacity for de novo lipid synthesis is a metabolic feature of most cancer cells that distinguishes them from their cells of origin. However, the mechanisms through which oncogenes alter lipid metabolism are poorly understood. We find that expression of oncogenic PI3K (H1047R) or K-Ras (G12V) in breast epithelial cells is sufficient to induce de novo lipogenesis, and this occurs through the convergent activation of the mechanistic target of rapamycin complex 1 (mTORC1) downstream of these common oncogenes. Oncogenic stimulation of mTORC1 signaling in this isogenic setting or a panel of eight breast cancer cell lines leads to activation of the sterol regulatory element-binding proteins (SREBP1 and SREBP2) that are required for oncogene-induced lipid synthesis. The SREBPs are also required for the growth factor-independent growth and proliferation of oncogene-expressing cells. Finally, we find that elevated mTORC1 signaling is associated with increased mRNA and protein levels of canonical SREBP targets in primary human breast cancer samples. These data suggest that the mTORC1/SREBP pathway is a major mechanism through which common oncogenic signaling events induce de novo lipid synthesis to promote aberrant growth and proliferation of cancer cells.Oncogene advance online publication, 1 June 2015; doi:10.1038/onc.2015.179.
... The 'total1 activity of PDH in the mammary gland of the lactating rat increases seven-to-ten fold from mid pregnancy to mid lactation (Gumaa et Nakanishi and Numa (1970) showed that the amount of immunotitratable ACC in rat liver is more than halved by a 48 hour fast and increased nearly 3-fold above controls when the animals were refed a fat free diet. This was due to simultaneous changes in the rate of degradation and synthesis of ACC. ...
Thesis
The mammary gland of the lactating rat uses 30 mmol of glucose a day. 70% of this is used for the synthesis of fatty acids which is therefore stringently regulated. This thesis describes investigations designed to elucidate some of the mechanisms of this regulation. Acetyl-CoA carboxylase (ACC) and pyruvate dehydrogenase (PDH) are key regulatory enzymes in the pathway from glucose to fatty acid. Pyruvate dehydrogenase catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA and ACC catalyses the committed step in the synthesis of fatty acids from acetyl-CoA. In lactating rat mammary gland the activities of both enzymes are profoundly inhibited by starvation and rapidly re-activated (within 3 hours) upon refeeding. Unexpectedly during these dietary manipulations the rate of fatty acid synthesis can be more closely correlated with PDH than ACC even though the latter is considered to be the major rate determining enzyme. Perfusion of the mammary gland in situ was performed using known inhibitors of fatty acid synthesis in the perfusate. Palmitic acid inhibited PDH but not ACC; acetoacetate had no effect on either enzyme. In vitro experiments suggest differential effects of palmitate (16:0) and oleate (18:1), palmitate being a less potent inhibitor of fatty acid synthesis and ACC than oleate. ACC in lactating rat mammary gland is phosphorylated and inactivated in response to 24hr starvation. One possible candidate for such phosphorylation is cAMP-dependent protein kinase. This had been purified from lactating mammary gland and its unusual tissue specific properties are described and discussed in relation to the results of in vivo experiments showing that in mammary gland the activity of this kinase does not correlate with the rate of fatty acid synthesis or ACC activity. The partial purification of a kinase capable of phosphorylating and inhibiting ACC in a manner identical to that which occurs during starvation in vivo is described. Its characteristics, regulation and physiological significance are discussed.
... The β-alanine, propanoate, pyruvate, fatty acid, unsaturated fatty acid and biotin metabolic pathways, which all significantly affect the synthesis of acetyl-coenzyme A and malonyl coenzyme A, were among the top 20 enriched pathways. Acetyl-coenzyme A is a pivotal product in the metabolism of energy substances in plants and the three major nutrients proteins, fats and sugars are aggregated into a common metabolic pathway [61]. Acetyl-coenzyme A is a precursor of synthetic ketone bodies, fatty acids, monoterpenoid alkaloids, steroids, carotenoids and so forth. ...
Article
Full-text available
Light is an important factor that affects the synthesis of functional metabolites in longan embryogenic calli (ECs). However, analysis of the effect of light on functional metabolites in longan ECs via RNA sequencing has rarely been reported and their light regulation network is unclear. The contents of various functional metabolites as well as the enzymatic activities of superoxide dismutase and peroxidase and the level of H2O2 in longan ECs were significantly higher under blue light treatment than under the other treatments (dark, white). In this study, we sequenced three mRNA libraries constructed from longan ECs subjected to different treatments. A total of 4463, 1639 and 1806 genes were differentially expressed in the dark versus blue (DB), dark versus white (DW) and white versus blue (WB) combinations, respectively. According to GO and KEGG analyses, most of the differentially expressed genes (DEGs) identified were involved in transmembrane transport, taurine and hypotaurine metabolism, calcium transport and so forth. Mapman analysis revealed that more DEGs were identified in each DB combination pathway than in DW combination pathways, indicating that blue light exerts a significantly stronger regulatory effect on longan EC metabolism than the other treatments. Based on previous research and transcriptome data mining, a blue light signaling network of genes that affect longan functional metabolites was constructed and HY5, PIF4 and MYC2 were shown to be the key regulatory genes in the network. The results of this study demonstrate that the expression levels of phase-specific genes vary with changes in longan EC functional metabolites.
... The acetyl-CoA carboxylase (ACC) activity was determined by [ 14 C] sodium bicarbonate incorporation leading to the formation of [ 14 C] malonyl-CoA as previosly described [34]. Briefly, the freeze-clamped liver were macerated in liquid nitrogen and eight volumes of a medium containg 0.1 M manitol, 50 mM sodium fluoride, 10 mM Tris, 1 mM EDTA, 5 mg·L −1 aproptin and 5 mg·L −1 leupeptin. ...
Article
Severe rheumatoid cachexia is associated with pronounced loss of muscle and fat mass in patients with advanced rheumatoid arthritis. This condition is associated with dyslipidemia and predisposition to cardiovascular diseases. Circulating levels of triglycerides (TG) and free fatty acids (FFA) have not yet been consistently defined in severe arthritis. Similarly, the metabolism of these lipids in the arthritic liver has not yet been clarified. Aiming at filling these gaps this study presents a characterization of the circulating lipid profile and of the fatty acids uptake and metabolism in perfused livers of rats with adjuvant-induced arthritis. The levels of TG and total cholesterol were reduced in both serum (10–20%) and liver (20–35%) of arthritic rats. The levels of circulating FFA were 40% higher in arthritic rats, possibly in consequence of cytokine-induced adipose tissue lipolysis. Hepatic uptake and oxidation of palmitic and oleic acids was higher in arthritic livers. The phenomenon results possibly from a more oxidized state of the arthritic liver. Indeed, NADPH/NADP⁺ and NADH/NAD⁺ ratios were 30% lower in arthritic livers, which additionally presented higher activities of the citric acid cycle driven by both endogenous and exogenous FFA. The lower levels of circulating and hepatic TG possibly are caused by an increased oxidation associated to a reduced synthesis of fatty acids in arthritic livers. These results reveal that the lipid hepatic metabolism in arthritic rats presents a strong catabolic tendency, a condition that should contribute to the marked cachexia described for arthritic rats and possibly for the severe rheumatoid arthritis.
... This increase in specific activity potentially could result either from an increased concentration of the enzyme in vivo or to a change in the catalytic efficiency of the molecules present. Similar variations in the levels of acetyl-CoA carboxylase activity have been induced in rat liver by altering the diet which the animals have been fed, and have been studied in some detail by Majerus and Kilburn [31] as well as by Nakanishi and Numa [32]. ...
Article
A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150000 g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein present in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mulants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Darkgrown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carhoxylasc activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.
Article
Acetyl-CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35–70-fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240000, containing 1.02 ± 0.04 molecules of biotin and 3.1 ± 1.7 molecules of alkali-labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [γ-³²P]ATP and magnesium ions with the purified catalytic subunit of cyclic-AMP-dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinase, and can be reversed by incubation with purified protein phosphatase-1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near-stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl-CoA carboxylase by protein phosphatase-1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333–338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic-AMP-dependent protein kinase or acetyl-CoA carboxylase kinase-2.
Article
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Withania coagulans (family: Solanaceae, English: Indian Cheese Maker, Hindi: Doda Paneer) fruit is known for its ethanopharmacological significance in health care system of India. Diet rich in high-fat is an important risk factor for diabetes, atherosclerosis and macro and microvascular complications. Treatment with aqueous extract of fruit of W. coagulans (aqWC; 250 mg/kg body weight) in cholesterol-fed animals resulted in significant decrease in the levels of total cholesterol, triacylglycerol, low density lipoprotein, tissue lipid content and acetyl CoA carboxylase activity whereas, the level of high density lipoprotein and activity of HMGCoA reductase also recovered partially. Treatment with aqWC also significantly decreased plasma lipid peroxide levels and increased reduced glutathione and superoxide dismutase activities. These results suggest that the aqueous extract of W. coagulans has potent lipid lowering and antioxidant activities.
Article
Rates of lipogenesis, as measured by the incorporation in vitvo of [14C]acetate into fatty acids, were measured in wethers which were either fed continuously or subjected to an interval of starvation. Refeeding ad libitum of starved sheep resulted in a faster growth rate; immature sheep after 110 days and mature sheep after 56 days had reached a fat content similar to that in the continuously grown sheep. In continuously grown sheep the rate of lipogenesis in subcutaneous adipose tissue increased with increasing liveweight. Lipogenesis was almost totally eliminated after a period of weight loss. In nutritionally rehabilitated immature and mature sheep, lipogenesis was restored and reached a rate higher than that measured for sheep which were continuously grown on the same diet. In immature sheep lipogenesis responded rapidly to refeeding. However, lipogenesis in mature sheep was slower to respond to increased dietary intake, which indicated that in mature sheep the biochemical pathways of lipid metabolism take several days to respond and reach their full lipogenic potential in response to changes in the physiological state.
Article
An aqueous methanol extract from green tea showed potent acetyl-CoA carboxylase inhibitory activity. An active compound was isolated from the extract and identified as (-)-epigallocatechin gallate by instrumental analyses, The IC50 value of (-)-epigallocatechin gallate was 3.1 x 10(-4) M. Among tea catechins and related compounds, nearly equal activity was found in(-)-epigallocatechin gallate and (-)epicatechin gallate, whereas (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, gallic acid and methyl gallate each had no inhibitory activity. These results indicate that the 3-O-gallate group of the catechin structure was necessary for this activity. (-)-Epigallocatechin gallate inhibited triglyceride accumulation in 3T3-L1 cells at a concentration of 1.0 x 10(-7) M or higher.
Article
Acyl-coenzyme-A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double-diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X-100 and chromatography on phosphocellulose, Blue-Sepharose and Sephadex G-100. The purified enzyme exhibits a specific activity of 20–24 U/mg protein at 25°C, which is about 100-fold higher than those of long-chain acyl-CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84000. The enzyme is specific for fatty acids with 14–18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl-CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl-CoA synthetase II.
Article
Present study aims to evaluate the effect of Withania coagulans fruit (aqWC) on diabetic-dyslipidemia and antioxidant/oxidant status in DM. Diabetic animals were treated with aqWC at a dose of 250mg/kg bw for 30days. Lipid profile, MDA, GSH, SOD, FRAP, HMG CoA reductase and acetyl CoA carboxylase activities were estimated in blood and tissues. Total cholesterol, TAG and LDL were significantly elevated whereas HDL was decreased in diabetic animals (p<0.05), simultaneously the lipid content and HMG CoA reductase activities were also increased, whereas acetyl CoA carboxylase activity decreased significantly in tissues of diabetic animals. MDA was increased and antioxidants such as SOD, GSH and FRAP decreased significantly in DM (p<0.05). Oral administration of aqWC to diabetic animals produced significant improvement in serum lipid profile and tissue lipid content. Activity of HMG CoA reductase decreased, whereas acetyl CoA carboxylase activity increased significantly in tissues after aqWC treatment. Administration of aqWC to diabetic animals also showed significant increase in antioxidant levels i.e., GSH, SOD, FRAP and reduced level of MDA in blood and tissue homogenates as compared to diabetic controls (p<0.05). These results suggest that aqWC treatment improved lipid profile and decreased oxidative stress in diabetes mellitus.
Article
This study was undertaken with growing rats to compare the effects of dietary 0.1% myo-inositol or 0.5% sodium phytate on the changes in the metabolism of hepatic lipids due to sucrose feeding. Liver weight, hepatic concentrations of total lipids and triglyceride, and hepatic activities of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), malic enzyme (ME, EC 1.1.1.40), fatty acid synthetase (FAS, EC 2.3.1.85), citrate cleavage enzyme (CCE, EC 4.1.3.8) and acetyl-CoA carboxylase (CBX, EC 6.4.1.2) were remarkably higher in rats fed on sucrose than in rats fed on corn starch for 12 or 13 days. Dietary myo-inositol and phytate significantly depressed the rises in liver weight, hepatic concentrations of total lipids and triglyceride, and hepatic activities of G6PD, ME, FAS and CCE due to sucrose feeding. These results suggested that dietary myo-inositol and phytate both can protect sucrose-fed animals against an accumulation of hepatic lipids, which might be at least in part ascribed to the depression in the hepatic lipogenesis.
Article
The effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-CoA carboxylase in rat liver were investigated following a cDNA cloning. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate was increased 2.5-fold in only 1 h. The mRNA concentration reached a maximal level of 9–12-fold increase in 8–16 h, and the enzyme induction increased 10-fold in 48 h. By a carbohydrate diet without protein, the transcriptional rate, mRNA concentration and enzyme induction were similarly increased to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce acetyl-CoA carboxylase. Corn oil feeding decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. After insulin treatment, the transcriptional rate was increased 2-fold (the normal level) in 2 h in diabetic rats. By fructose feeding to diabetic rats, the transcriptional rate and mRNA concentration were increased similarly to the levels reached by insulin treatment, while the enzyme induction was increased by only 60%. Thus, it is suggested that insulin is importantly involved in the transcription and also translation of acetyl-CoA carboxylase. On the other hand, triiodothyronine treatment increased the mRNA and enzyme levels in diabetic and normal rats, and somewhat increased the transcriptional rate only in diabetic rats. Triiodothyronine appears to stabilize the mRNA besides having an insulin-like action in acetyl-CoA carboxylase transcription.
Article
The purposes of this study were to identify exogenous factors that would depress synthesis of saturated fats and enhance synthesis of unsaturated fats in the dairy cow’s mammary gland. Certain long-chain exogenous fatty acids are known to modulate endogenous fat synthesis within tissues. We analyzed the effects of two different long-chain monounsaturated fatty acids, namely oleic acid and trans-vaccenic acid (TVA), on activities of acetyl-CoA carboxylase (ACC), fatty acid synthetase (FAS) and stearoyl-CoA desaturase (SCD) in bovine mammary epithelial cell cultures. The study was done using an established bovine mammary epithelial cell line, the MacT cells. ACC (EC 6.4.1.2) and FAS (EC 2.3.1.85) are two major enzymes involved in biosynthesis of saturated fatty acids in eucaryotic cells. SCD (EC 1.14.99.5) is the enzyme catalyzing the critical committed step in biosynthesis of unsaturated fatty acids from their saturated precursors. Data indicated depression of activity of enzymes responsible for mammary synthesis of saturated fatty acids (ACC and FAS), along with a simultaneous enhancement of mammary desaturase activity, by TVA.
Article
The rates of synthesis and degradation of glucose-6-phosphate dehydrogenase in the liver of male C57BL mice are followed by a modified immunochemical method. Mice are given labeled amino acids for a predetermined period, and the labeled enzyme is isolated from liver homogenates with goat anti-enzyme, followed by rabbit anti-goat IgG serum. The precipitates are either counted directly or analyzed on acrylamide gels. The rate of glucose-6-phosphate dehydrogenase synthesis is found to be independent of the state of induction or repression of the animal. The synthesis of the enzyme constitutes 0.20–0.25% of the synthesis of all soluble liver proteins. This rate is maintained even during periods of most rapid formation or disappearance of enzyme activity. Quantitative immunoprecipitation shows that similar amounts of precipitable antigen are present in induced and non-induced liver homogenates. The results indicate that the large reversible increase in activity of glucose-6-phosphate dehydrogenase, observed when animals are transferred from a high-fat to a fatless diet, does not involve the synthesis of a new enzyme protein, and that mechanisms involving modulation of existing enzyme molecules ought to be considered for the adaptation of this enzyme in mouse liver.
Article
This chapter describes the determination of acetyl coenzyme A carboxylase from rat liver. In the course of assay, [14C]bicarbonate is converted into the carboxyl group of malonyl-CoA. As a result an acid-volatile compound is converted into an acid-stable compound. The reaction is stopped by addition of acid, and the reaction mixture is taken to dryness. Unreacted [14C] bicarbonate escapes as 14CO2, while the 14C fixed into malonyl-CoA remains. The radioactivity of the residue is a measure of the activity of the enzyme. This assay gives linear results only during the initial portion of the reaction because the accumulation of malonyl-CoA causes the reaction to become inhibited. A difficulty is encountered, when this assay is applied to crude extracts because such extracts usually contain the enzymes and substrates for other CO2-fixing reactions. Even when all the substrates needed for such reactions are not present, exchange reactions can still cause an unwanted CO2 fixation. This difficulty is readily overcome by subjecting the crude extracts to gel filtration on Sephadex G-25 prior to assay. In crude extracts, acetyl-CoA carboxylase from rat must be activated by incubation with citrate or, better, with citrate and magnesium ions, before it can be assayed under conditions of maximum activity. This type of activation is inhibited by adenosine triphosphate (ATP).
Thesis
p>In situ fatty acid synthesis has been measured with 3H20 in lean and obese Zucker (fa/fa) rats. The accumulation of fatty acids was increased in both the liver and adipose tissue of young fa/fa rats as a result of both an increased rate of lipogenesis and an increase in tissue mass. Whereas total hepatic lipogenesis increased with age, total adipose tissue lipogenesis decreased in older fa/fa rats. Experiments with hepatectomized rats showed that the liver was the major site of the excess fatty acid synthesis in the fa/fa rats. The enhanced rate of lipogenesis in fa/fa rats was abolished by either pair feeding or streptozotocin treatment. The results suggest that the increased fatty acid synthesis in fa/fa rats is secondary to the hyperphagin, hyperinsulinaemia and increased mass of hepatic and adipose tissues. Sucrose feeding resulted in an increased hepatic and adipose tissue lipogenesis and in insulin levels in lean animals with an increase in hepatic lipogenesis and insulin levels in the fa/fa rats. Adrenalectomy decreased the rates of lipogenesis in both the liver and adipose tissues, insulin levels and weight gain of fa/fa rats.Pre-obese fatty rats have been identified by their lower rectal temperature (34.6 + 0.2°C v 35.4 + 0.3°C) from day 16 onwards. Hepatic lipogenesis, hepatic glucose-6-phosphate dehydrogenase, hepatic acetylCoA carboxylase and insulin levels remained unchanged in suckling pre-obese rats and increased only after weaning when all the values for the obese were significantly different from values obtained for lean rats. However, adipose tissue lipogenesis, glucose-6-phosphate dehydrogenase, acetylCoA-carboxylase and adipocyte size were all significantly increased in.the pre-obese suckling rats from day 10 onwards. The results suggest that the primary genetic defect in the fatty rats may be related to either a defective thermogenic process or a defective control of adipose tissue lipogenesis.</p
Chapter
This chapter discusses the catalytic reactions and molecular characteristics of acyl-CoA carboxylases. Acyl-CoA carboxylases [acyl-CoA: carbon dioxide ligases (ADP)] are enzymes containing covalently bound biotin that catalyze an ATP- dependent carboxylation of an acyl-CoA. Three enzymes of this type have been intensively studied—β-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and acetyl-CoA carboxylase. One or all of these enzymes play central roles in the metabolism of most of the life forms. β-Methylcrotonyl-CoA carboxylase catalyzes the biotin-dependent carboxylation of P-methylcrotonyl-CoA to form p-methylglutaconyl- CoA in a two-step reaction. The discovery that biotin is involved in leucine metabolism and that it participates directly in the p-methylcrotonyl-CoA carboxylase reaction suggested that P-methylcrotonyl-CoA or some earlier metabolites, or metabolite derivatives of this pathway might accumulate in biotin deficiency states. β-methylcrotonyl-CoA carboxylase catalyzes the carboxylation of β-methylcrotonyl-CoA to form β-methylglutaconyl-CoA. Propionyl-CoA carboxylase catalyzes the cmboxylation of propionyl-CoA to form S-methylmalonyl-CoA. The apparent substrate affinity constants for the enzymes isolated from both the pig heart and the bovine liver mitochondria are essentially identical.
Thesis
Viable, rod-shaped, trypan blue-exclusive cardiac myocytes were purified from collagenase perfused heart. The cells showed typical cross-striations and rhythmical beating movements. This ceil preparation was used to demonstrate the existence of the soluble enzymes ATP-citrate lyase and a citrate-dependent acetyl-CoA carboxylase, together with the membrane-bound condensing enzyme of the fatty acid elongation system. The Km for malonyl-CoA of the fatty acid elongation system was 50μM. Using sensitive radiochemical assay techniques, no fatty acid synthase activity was demonstrable in myocytes. Significant levels of the enzyme were measureable in extracts of whole heart. The myocytes were also used to investigate the carbohydrate-mediated suppression of fatty acid oxidation. Using either 5mM glucose or 2mM lactate, the rate of [1-14C]paimitate was decreased by approximately 40%. In the presence of glucose, insulin (8nM) decreased and adrenaline (5μM) increased the rate of palmitate oxidation by myocytes (30% and 100% respectively). With glucose present 5μM phenylephrine decreased palmitate oxidation by 23%, and 5μM isoprenaline increased palmitate oxidation by 40%. Insulin and adrenaline had no significant effect in the absence of glucose. The levels of malonyl-CoA, long-chain acyl-CoA and carnitine esters were measured from freeze-clamped, non-working perfused hearts. In the presence of glucose, malonyl-CoA content was measured at 4.6μM. Insulin acutely raised this level by 34%. With insulin present, both palmitate (0.5mM) and adrenaline (5μM) decreased the malonyl-CoA content by 60% and 46% respectively. Insulin had no significant effect on the size of the combined pool of long-chain fatty acyl CoA and carnitine esters. Perfusion with palmitate significantly increased the content of both long-chain esters but decreased the ratio of acylcarnitine/acyl-CoA. Perfusion with adrenaline also significantly increased the content of both long-chain esters, in particular acylcarnitine. Adrenaline increased the content of the total long-chain ester pool by at least as much as palmitate.
Article
Acetyl coenzyme A (acetyl-CoA) carboxylase activity, amount, and mRNA levels increase during the differentiation of 30A-5 preadipocytes to adipocytes. Tumor necrosis factor (TNF) completely prevents this differentiation, with concomitant inhibition of acetyl-CoA carboxylase mRNA accumulation. To investigate the mechanisms by which TNF prevents acetyl-CoA carboxylase mRNA accumulation, we determined the effect of TNF on the transcription rate of the carboxylase gene and the half-life of carboxylase mRNA. Nuclear runoff transcription assays revealed no differences in the number of RNA polymerase molecules actively engaged in transcription of the acetyl-CoA carboxylase gene in preadipocytes, adipocytes, TNF-treated preadipocytes, or at any time during the course of differentiation. However, changes in adipsin, glycerophosphate dehydrogenase, and actin mRNAs, whose levels are also differentiation dependent, can be accounted for in part by changes in the number of polymerase complexes on their respective genes. To determine whether TNF caused a decrease in the stability of carboxylase RNA transcripts, we measured the rate of decay of prelabeled acetyl-CoA carboxylase mRNA. Control and TNF-treated cells showed no difference between the apparent half-lives of acetyl-CoA carboxylase mRNAs (9 h). However, the rate of acetyl-CoA carboxylase mRNA synthesis in vivo was decreased three- to fourfold in the presence of TNF. These data demonstrate that TNF prevents accumulation of acetyl-CoA carboxylase mRNA during preadipocyte differentiation by decreasing the rate of acetyl-CoA carboxylase gene transcription. However, transcriptional control is not due to a change in the number of RNA polymerase complexes actively engaged in carboxylase transcript elongation which could be measured by a number runoff assay. Instead, transcriptional control may be related to the rate at which RNA polymerase traverses the acetyl-CoA carboxylase gene.
Article
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The in vitro inhibition of hepatic acetyl-CoA carboxylase by lipid-lowering agents, reported previously, is also demonstrable in drug-treated rats. Levels of measurable acetyl-CoA carboxylase activity are significantly lower in the livers of rats treated with 2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl)-phenoxy]-propionic acid than those of controls animals. Male or female and different strains of rats exhibit this depression of enzyme activity on drug treatment under a variety of nutritional states, including pair feeding. The inhibition is reversible, as shown by annulment of the effect on dialysis of the enzyme preparations against a citrate-containing buffer. Treatment of rats with ¹⁴C-labeled drug and fractionation of the liver homogenate shows preferential association of the drug with the acetyl-CoA carboxylase-containing fraction. On dialysis the ¹⁴C activity of the drug is removed and the enzyme activity returns to control levels.
Article
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The short term regulation of fatty acid biosynthesis has been studied using primary cultures of human skin fibroblasts. These cells show rapid changes in rates of acetate incorporation into fatty acids depending upon the lipid content of the medium. When cells are incubated in medium containing lipid-deficient fetal calf serum overnight and then are switched to Eagle's minimum essential medium, the rate of acetate incorporation into fatty acids is unaffected. Addition of 5 µm albumin-bound palmitic, stearic, oleic, linoleic, or arachadonic acids to the minimum essential medium results in a 26 to 67% inhibition in the rate of acetate incorporation depending upon the fatty acid used. These results are obtained when cells are incubated with [1-¹⁴C]acetate from 0.5 to 10.5 min after addition of fatty acid to the medium. When cells are labeled from 5 to 15 min after addition of fatty acid, there was a greater inhibition of acetate incorporation to 70 to 85%. Several lines of evidence suggest that the site of action of fatty acids is intracellular. The effect of added fatty acids on the intracellular concentration of citrate, long chain acyl-CoA compounds, and free fatty acids were measured in these cells. Long chain acyl-CoA levels and citrate levels of cells incubated in Eagle's minimum essential medium for 10.5 min are 73.3 ± 2.5 pmoles per mg of total cellular protein and 2.1 ± 0.3 nmoles per mg of total cellular protein, respectively. When albumin-bound palmitic acid at a concentration of 2.1 µm is included in the incubation medium the levels of long chain acyl-CoA compounds (69.0 ± 3.0 pmoles per mg of total cellular protein) and of citrate (1.9 ± 0.1 nmoles per mg of total cellular protein) remain unchanged despite inhibition of acetate incorporation into fatty acid by this time. Furthermore, incubation of cells in medium containing albumin-bound palmitic acid results in only slight increases in the intracellular free fatty acid levels compared to cells incubated in fatty acid-free medium. These results suggest that total cellular levels of these metabolites are not directly responsible for regulation of the rate of acetate incorporation into fatty acids.
Article
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Acetyl-CoA carboxylase from rat liver has been purified to homogeneity as judged by gel electrophoresis, ultracentrifugation, and ultracentrifugation in the presence of 6 m guanidine hydrochloride. When the enzyme is subjected to gel electrophoresis in the presence of sodium dodecyl sulfate a sharp protein band is observed which has a molecular weight of about 215,000. In addition two sharp bands are observed which run closely together and which have molecular weights of about 118,000 and 125,000. Prolonged or repeated treatment of the heavy band with sodium dodecyl sulfate at an elevated temperature results in its conversion to the two light bands. It is concluded that the enzyme contains pairs of unlike subunits which remain associated to a considerable degree in sodium dodecyl sulfate at room temperature but which can be separated after more drastic treatment. Ultracentrifugation of the native enzyme yields an s20,w value of 45. Molecular weight determinations by sedimentation equilibrium in the presence of 6 m guanidine hydrochloride and mercaptoethanol yield a molecular weight for the subunits of the enzyme of 111,100. The amino acid composition, biotin content, and phosphate content of the enzyme are presented.
Article
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Partially purified acetyl-CoA carboxylase (EC 6.4.1.2) was activated several-fold by incubation with magnesium ions. The activation was inhibited by fluoride. Partially purified but not highly purified enzyme was inactivated by ATP in a reaction which was both time- and temperature-dependent. Inactivation by ATP was greatly enhanced by a protein fraction devoid of carboxylase activity, and the inactivation was independent of adenosine 3′,5′-monophosphate. Whereas inactivation of the enzyme in the presence of [γ-³²P] ATP resulted in significant amounts of protein-bound ³²P, incubation with [U-¹⁴C]ATP led to little incorporation of isotope into protein. The ³²P label accompanied the carboxylase activity through ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The ³²P-labeled carboxylase was precipitated by the antibody to homogeneous acetyl-CoA carboxylase. The evidence is consistent with a mechanism for controlling acetyl-CoA carboxylase activity by an interconversion involving phosphorylation and dephosphorylation reactions. It appears that the carboxylase protein is phosphorylated, and the activity decreased, by an ATP-dependent kinase. It is reactivated by dephosphorylation that is mediated by a magnesium-dependent phosphatase.
Article
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Immunochemical techniques have been utilized to study the content, synthesis, and degradation of the fatty acid synthetase in liver and brain during development and in various nutritional states. The distinctive changes in synthetase activity during development of both tissues are related entirely to changes in content of enzyme. During fasting and fat-free feeding, in contrast to the lack of change in synthetase activity of brain, there are dramatic alterations in hepatic activity. These changes are related entirely to changes in content of enzyme in liver. During development of brain the rate of synthesis of the enzyme decreases. In developing liver, when synthetase activity rises at the time of weaning, there is a marked acceleration of synthesis of enzyme. During fasting, when hepatic synthetase concentration falls drastically, there is a considerable reduction in synthesis of enzyme, and when animals are refed a fat-free diet, a remarkable increase in enzyme synthesis occurs, confirming earlier reports. The rate of fatty acid synthetase degradation in brain of adult animals differs considerably from that in liver; the half-lives are 6.4 and 2.8 days, respectively. In young suckling animals the rate of degradation of the enzyme in brain is markedly accelerated (t1/2 = 1.9 days), whereas in liver there is little change (t1/2 = 2.3 days). The most profound change in degradation of synthetase in liver occurs during fasting when the t1/2 decreases to 18 hours. No changes were observed in degradation in brain during fasting or fat-free feeding. Rate constants for synthesis and degradation of fatty acid synthetase in liver and brain during development and in the various nutritional states were derived; these quantitatively accounted for the differences in content of the enzyme in these tissues. In developing liver the dramatic elevation in synthetase content apparent after weaning is associated with an acceleration of enzyme synthesis but no change in rate of enzyme degradation. The changes in enzyme content during development of brain are accompanied by alterations in the rates of synthesis and degradation of fatty acid synthetase. Both processes are more rapid in the brain of the young suckling animal than in the mature animal. These observations represent the first direct measurements of rates of synthesis and degradation of an enzyme in mammalian brain.
Thesis
Malonyl-CoA plays a role in the regulation of cardiac β-oxidation due to its ability to inhibit carnitine palmitoyltransferase 1. Acetyl-CoA carboxylase (ACC) is the enzyme responsible for the synthesis of malonyl-CoA, the concentration of which increases in hearts exposed to glucose and insulin and is decreased by physiological concentrations of fatty acids and by adrenaline. ACC is phosphorylated and inhibited in response to the activation of the AMP- activated protein kinase (AMPK) cascade. Both α-1 and α-2 AMPK isoforms are phosphorylated and activated in hearts perfused with physiological concentrations of palmitate. This activation is not associated with alterations in the ratio of AMP to ATP. The activity of both AMPK isoforms is decreased after exposure to insulin, an effect overcome by 0.5mM palmitate. The activity of α-1 AMPK is stimulated in glucose perfused hearts by adrenaline, with rates similar to those measured in hearts perfused with 0.5mM palmitate. Adrenaline failed to increase α-1 AMPK activity above that due to fatty acids in palmitate perfused hearts. Adrenaline stimulation of palmitate perfused hearts resulted in a reversal of the effect of this fatty acid on α-2 AMPK activity. Adrenaline was without effect on the activity of this isoform in hearts perfused with glucose as the sole substrate. ACC was highly phosphorylated at the AMPK site in tissue from hearts exposed to 0.5mM palmitate compared to those perfused with glucose alone. However despite this, no alteration in ACC activity could be measured in response to palmitate and hormones in this study except for a small decrease due to adrenaline in palmitate perfused hearts.
Article
We investigated the changes in triglyceride metabolism induced by various lipid loading in aged and young rats in order to elucidate the mechanism of hyperlipidemia in the aged. 24 month-old rats were used as the aged and were compared with 2 month-old rats. Serum triglyceride in the aged rats were higher than those of the young rats. After oral oil loading, serum triglyceride was increased more accurately in aged rats than in young rats. There are three possible mechanisms by which serum triglyceride was increased. (1) Increase in triglyceride absorption from intestine. (2) Increase in triglyceride synthesis in liver. (3) Decrease in triglyceride clearance from serum. As to the possibility of (1), it may be excluded because decrease in triglyceride absorption from intestine according to aging was reported. As to the possibility of (2), activities of Acetyl-CoA carboxylase, Acyl-CoA synthetase and Triglyceride Synthesis were measured in the liver of the aged or the young rats. These three activities were not different in the aged and the young rats. As to the possibility of (3), decrease in triglyceride clearance from serum was observed in the aged rats after intravenous administration of lipid. One of the most important factors which regulate triglyceride clearance from serum is lipoprotein lipase activity in vascular wall. We discussed the capacity of this enzymatical activity by measuring post heparin lipolytic activity (PHLA) and heparin releasable lipoprotein lipase activity in epididymal adipose tissue in the aged or the young rats. The both activities were decreased in the aged rats compared with the young rats. Above results suggest that hypertriglyceridemia in the aged might depend on the decrease in the capacity or adaptability of lipoprotein lipase activity to postprandial increase in serum lipid.
Chapter
The de novo synthesis of saturated fatty acids, studied in a variety of biological systems, is catalyzed by two enzyme systems, acetyl-CoA carboxylase and fatty acid synthetase. These enzyme complexes are found in the cell cytoplasm when cells are ruptured and the cellular components are fractionated by usual techniques. All the carbon atoms of the fatty acids are derived from acetyl-CoA. Although palmitate is the major fatty acid that is produced by most biosynthetic systems, the chain length of the fatty acid that is produced varies in different biological sources and under different experimental conditions. Examination of the substrates of fatty acid synthesis, acetyl-CoA, adenosine triphosphate (ATP), and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), suggests that this biosynthetic pathway can be controlled by regulation of the availability of these substrates. Acetyl-CoA carboxylase catalyzes the first reaction in the synthesis of fatty acids, which is unique to this biosynthetic pathway, and, therefore, this enzyme is regulated.
Chapter
The turnover characteristics (synthesis and degradation) of lipogenic enzymes in differentiating cells have been compared with respect to the nature of the lipogenic stimuli and the turnover response of the enzymes. In particular studies are described on systems where detailed measurements of changes in enzyme amount, rate of synthesis and rate of degradation have been made. Where possible the data has been analysed by a computer program which can calculate changes in any one of these parameters given measurements of the other two. Transitory changes in the rate of enzyme degradation have been determined from measured increases in the amount and rate of synthesis of an enzyme after a differentiation stimulus. The computations have been considered in relation to a model which examines the putative role of transitory changes in enzyme degradation as part of a regulated program of enzyme turnover during cell differentiation.
Chapter
Neutral lipids, especially triacylglycerols, accumulated due to myo-inositol deficiency both in the cells of Saccharomyces carlsbergensis and in the liver of rats. The mechanisms were investigated.
Chapter
In spite of the wide-spread occurrence of cell death in plants, the phenomenon is a topic rarely discussed in textbooks of plant anatomy or physiology. It is implicated extensively in the death of whole plants, e. g. monocarpic plants which reproduce once only and die, or polycarpic plants which reproduce either a few or many times before dying, and with the death of parts of polycarpic plants. This latter point is seen in the case of flower, leaf and fruit fall, bark-shedding or with the loss of the shoot after reproduction in some herbaceous plants. In addition, cell death is involved in both morphogenesis and a number of normal physiological events in cryptogams and phanerogams (Table 5.1). It is the aim of this chapter to consider the possible mechanisms leading to cell death, the involvement of cell death in the various, normal events in plants, and the ways in which cell death might be determined in various cell populations. Many of the examples quoted will be from flowering plants, since it is in this domain that most work has been performed. Space will not permit an extensive treatment of the literature and, instead, as many and varied examples as possible will be used to illustrate the points under discussion.
Chapter
The integration of lipid metabolism is maintained predominantly by the liver. Excessive dietary glucose can be converted to fatty acids that are esterifled and exported in triglyceride-rich very-low-density lipoproteins. Excessive fatty acids provided by lipolysis in adipose tissue are either reesterified or converted to ketones via β-oxidation in the liver. Cholesterol is transferred by lipoproteins from peripheral organs to the liver for excretion into the bile. This is the main mechanism by which cholesterol is eliminated. The chapter reviews the pathways of lipid metabolism in the liver, including interorgan relationships and lipoprotein turnover. The short- and long-term regulation by substrates and hormones is described along with aspects of the zonal heterogeneity of lipid metabolism.
Article
There are doubts about the impact of non-nutritive sweeteners consumption on lipogenic and glycolytic metabolism. Therefore, the objective was to determine the effects of chronic consumption of sweeteners on the activity levels of the enzymes glucokinase (GK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PKL), acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) in livers’ extracts. Groups of male and female Wistar rats drank solutions of sweeteners for 480 days: Sucrose 10%, glucose 14%, fructose 7%, acesulfame K 0.05%, aspartame:acesulfame mixture 1.55%, sucralose 0.017%, saccharin 0.033%, and a control group. The enzymatic activity in livers’ extracts was determined. Likewise, the levels of glucose, triglycerides, insulin, glucagon, and leptin were determined. In both genders, there were significant differences in the levels of enzymatic activity, hormonal, and biochemical parameters due to sweeteners consumption. The highest glycolytic and lipogenic enzyme activity levels were observed in the groups that ingested nutritive sweeteners and saccharin.
Article
This chapter discusses the biosynthesis of saturated fatty acids. The de novo synthesis of saturated fatty acids, studied in a variety of biological systems, is catalyzed by two enzyme systems that function sequentially, acetyl-CoA carboxylase and fatty acid synthetase. These enzyme complexes are found in the cell cytoplasm when cells are ruptured and the cellular components are fractionated by usual techniques. The mechanism of action of acetyl-CoA carboxylase demonstrated that the enzymes contain covalently bound biotin as a prosthetic group and that a carboxybiotin intermediate is formed during the reaction. The mode of binding of the carboxyl group to the biotin was demonstrated by studying a model reaction, the carboxylation of free biotin by β-methylcrotonyl-CoA carboxylase; the product of this reaction was methylated and identified as 1'-N-carboxymethylbiotin methyl ester. Acetyl-CoA carboxylase is the first committed reaction in the biosynthesis of fatty acids, and this enzyme is known to be subject to two types of control, allosteric regulation and adaptive changes in enzyme content.
Chapter
The living organism needs fatty acids for the hydrophobic parts of biological membranes or as an energy store in the form of triglycerides. The requirement for fatty acids can be met either by biosynthesis or by dietary supply. Because fatty acids are essential for the proper functioning of the living organism, their synthesis and degradation must be precisely regulated so as to respond to various metabolic conditions. The first step in the pathway of long-chain fatty acid biosynthesis is mediated by acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), a biotin-containing enzyme which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, the activated donor of 2-carbon units for the elongation of fatty acids catalyzed by fatty acid synthetase. Because malonyl-CoA has no apparent metabolic alternative, it would be of teleonomic significance to regulate fatty acid synthesis at this carboxylation step. In fact, accumulated evidence indicates that acetyl-CoA carboxylase plays a critical role in the regulation of this biosynthetic process. The cellular content of the enzyme varies with the rate of fatty acid synthesis in different nutritional, hormonal, developmental and genetic conditions, and the catalytic activity of the enzyme is modulated by a number of metabolites and by phosphorylation/dephosphorylation of the enzyme. This chapter deals with the enzyme from animals, yeasts and bacteria.
Article
Kinetoc studies were carried out on the reaction mechanism of rat liver acetyl coenzyme A carboxylase. The results of initial velocity and product inhibition studies on the forward and reverse reaction indicate that the carboxylase reaction is most likely to proceed through the “bi bi uni uni ping pong” mechanism. The order of the addition of substrates to the enzyme is as follos: ATP, HCO3- and acetyl-CoA in the forward reaction, and malonyl-CoA, Pi and ADP in the reverse reaction. Kinetic analysis of the stimulatory effect of citrate on the initial velocities of the forward and reverse reaction indicate that the site of citrate action during catalysis lies predominantly onthe earboxylated enzyme. It is proposed that there exists and equilibrium between active and inactive forms of the carboxybiotinyl enzyme and that this equilibrium is shifted toward the active form in the presence of citrate.
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The factors effecting the reversible interconversion between the protomeric and polymeric forms of liver acetyl coenzyme A carboxylase were investigated by sucrose density gradient centrifugation and electron microscopy. Certain anions (citrate, isocitrate, malonate, tricarballylate, sulfate, and Pi), acetyl-CoA, high protein concentration, and pH 6 to 7 promote aggregation of the protomer. Other factors, such as carboxylation of the enzyme to produce enzyme-CO2⁻, Cl⁻, and pH values greater than 7.5, cause dissociation of the polymeric form. Citrate and isocitrate, but not tricarballylate or Pi, are capable of promoting the transition of the carboxylated enzyme (enzyme-CO2⁻) to the polymeric state. Under the conditions for carboxylase assay or for measuring the rates of other carboxylase-catalyzed reactions, all of which involve enzyme-CO2⁻ as intermediate, isocitrate and citrate promote aggregation of the carboxylase to its polymeric form and a 15- to 16-fold activation of catalysis; neither tricarballylate nor Pi has this effect. Binding experiments reveal that acetyl-CoA carboxylase has one tight binding site per protomer (mol wt 410,000) for citrate (KD, 2 to 3 × 10⁻⁶M) and another for acetyl-CoA (KD, 4 × 10⁻⁶M). Acetyl-CoA binding at the latter site is unaffected by the presence of citrate. Dissociation of acetyl-CoA carboxylase with sodium dodecyl sulfate (SDS; 0.1%) gives rise to subunits with a sedimentation coefficient (s20, w) of 4.3 S. By equilibrium dialysis against 0.1% SDS-³⁵S, the carboxylase was found to bind 0.57 g of the detergent per g of carboxylase protein. The subunit molecular weight, determined by sedimentation equilibrium corrected for detergent binding, was 114,000 and by electrophoresis in SDS-polyacrylamide gels (1% SDS), 110,000. The presence of nonidentical subunits is indicated by the fact that there is a single biotinyl prosthetic group and there are single binding sites for both citrate and acetyl-CoA on the 410,000 molecular weight protomer.
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The turnover of the protein constituents of the endoplasmic reticulum and certain cell fractions of rat liver has been examined by following the decay in specific radioactivity after a single administration of either uniformly labeled ¹⁴C-L-arginine or ¹⁴C-guanidino-L-arginine. Significantly shorter half-lives were obtained for all cell fractions when proteins were pulse-labeled with the guanidino-labeled arginine. The mean half-life for both rough and smooth microsomal membrane fractions was 2.0 to 2.1 days with guanidino-labeled arginine compared to 5.2 to 5.8 days with uniformly labeled arginine. We ascribe such differences to differing degrees of isotope reutilization. A new technique using two administrations of the same amino acid but labeled with different isotopes was developed to examine relative rates of degradation of proteins of the microsomal membranes. Proteins of the membrane, including NADPH-cytochrome c reductase, cytochrome b5, and protein fractions solubilized by detergent and separated on diethylaminoethyl-cellulose, had different rates of degradation. The administration of phenobarbital to rats induces proliferation of endoplasmic reticulum in the liver. Phenobarbital increased the rate of synthesis of some but not all proteins of the endoplasmic reticulum. The rate of synthesis of NADPH-cytochrome c reductase was increased 3- to 4-fold over the basal rate; the rate of cytochrome b5 synthesis was increased to an insignificant extent. The rate of synthesis of some membrane proteins was decreased during phenobarbital administration. Phenobarbital had no effect on the rate of degradation of total membrane protein. These results indicate that the endoplasmic reticulum is a dynamic system with a rapid rate of renewal and are consistent with a mosaic model of membrane biogenesis.
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Acetyl coenzyme A carboxylase has been isolated from chicken liver and purified more than 1000-fold in good yield. It is an unusually stable protein exhibiting negligible loss of enzymatic activity during 1 week of storage (1 to 5 mg of protein per ml) at room temperature in potassium phosphate buffer, pH 7. The pure enzyme catalyzes the carboxylation of 8.8 µmoles of acetyl-CoA (or 7.0 µmoles of propionyl-CoA) per min per mg of refractometrically determined protein at its pH optimum of 7.5 at 37°. The carboxylase-catalyzed carboxylation of acetyl-CoA is activated 15- to 16-fold by DL-isocitrate or citrate and 5-fold by malonate; tricarballylate is essentially inactive. Arrhenius plots for acetyl-CoA carboxylation in the presence or absence of isocitrate are biphasic, having a transition point at about 21°. Below this temperature, where there is very little activator effect, the temperature coefficient (Q10) in the presence of isocitrate is 8.0. Above the transition point, where isocitrate activation is evident, the temperature coefficient (Q10) in the presence of activator is 2.0 and in its absence is a negative value. At pH 7.5 and 37°, the carboxylase catalyzes the over-all reverse reaction (decarboxylation), ATP-³²Pi exchange, malonyl-CoA-¹⁴C-acetyl-CoA exchange, and ADP-, Pi-, and Mg²⁺-independent malonyl-CoA decarboxylation reactions at 14%, 6.2%, 33%, and 1.2%, respectively, of the rate of the over-all forward (carboxylation) reaction. All of the above mentioned reactions exhibit essentially the same tri- and dicarboxylic acid activation patterns. Evidence is presented which indicates that isocitrate activation of the carboxylase is associated with an increased maximum velocity.
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The roles of synthesis and degradation in the regulation of acetyl coenzyme A carboxylase levels in rat liver following fasting and fat-free feeding have been studied. Antibodies prepared to homogeneous chicken liver acetyl-CoA carboxylase were shown to cross-react with rat liver acetyl-CoA carboxylase. Enzyme from both species was totally inactivated as well as precipitated by this antibody preparation. Furthermore, quantitative precipitin curves and equivalence point determinations indicated that enzymes from the two species were precipitated in equal quantity by antibody and that the two enzymes had identical turnover numbers. It was also shown that disaggregated, aggregated, and palmityl-CoA-treated enzyme were all equally precipitated by antibody. Immunological analysis of crude homogenates of rat liver from animals treated with different diets, which had specific activities for acetyl-CoA carboxylase varying over 25-fold indicated that there was a constant amount of immunologically precipitable enzyme per unit of enzyme activity. The changes in acetyl-CoA carboxylase activity measured after dietary alteration result from changes in the enzyme content of liver rather than from activation or inhibition of preformed enzyme. The relative rates of acetyl-CoA carboxylase synthesis were determined by quantitative precipitation of the enzyme by antibody after pulse labeling with 3H-leucine. There was a 5- to 10-fold increase in the rate of enzyme synthesis after fat-free feeding of previously fasted rats. The rate of degradation of acetyl-CoA carboxylase in rats fed a fat-free diet was found to be first order with a t½ of approximately 48 hours, whereas in fasted rats a t½ of 18 hours was obtained. In rats fed a 12% fat diet the rate of acetyl-CoA carboxylase degradation was similar to that found in animals fed a fat-free diet, whereas the rate of synthesis was diminished, thus suggesting that independent factors regulate the rates of acetyl-CoA carboxylase synthesis and degradation in rat liver.
Article
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It was previously reported that Selenomonas ruminantium required one of the normal-chain fatty acids with 3–10 carbon atoms, when the cells were grown in a glucose medium. In order to investigate this phenomenon with a cell-free system, an enzyme preparation catalyzing incorporation of [2-14C]malonyl-CoA into long-chain fatty acids was obtained from this organism grown on glucose and n-valerate by sonic extraction and (NH4)2SO4 fractionation (55–90% satn.). This enzyme system required, in addition to malonyl-CoA and either NADH or NADPH, a medium-chain fatty acyl-CoA derivative. The effects of C2-C8 acyl-CoA derivatives in concentrations ranging from 10 to 80 μM. were compared and found to decrease in the following order: C6 > C7 > C8 > C5 > C4; propionyl-CoA and acetyl-CoA were practically ineffective. The difference in the effects of C5-C8 acyl-CoA derivatives appeared to be due mainly to different Km values for these acyl-CoA derivatives. A similar medium-chain acyl-CoA requirement was observed also for enzyme systems from the same organism grown on lactate and from Clostridium acetobutyricum, although these cells did not require fatty acids for growth.
Article
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Refeeding starved rats a fat-free diet over a 48 hr period brings about a marked elevation in the activity of the enzymes in liver cytoplasm which catalyze the synthesis of saturated fatty acids from acetyl CoA and malonyl CoA. Acetate incorporation into palmitoleic and oleic acid is also accelerated during this period. Enhanced capability for the synthesis of these fatty acids is reflected in the net accumulation of saturated and monounsaturated fatty acids, as well as the triglyceride fraction of the liver lipids. Coincident with these events the relative amount of linoleic acid among liver fatty acids rapidly falls. These changes are substantially the same as those observed in early linoleic acid deficiency.
Article
The carboxylation of acetyl-CoA to form malonybCoA is the first and ratelimiting step in the biosynthetic sequence leading specifically to long-chain fatty acids. Studies in greater & tall on acetyl-CoA carboxylase which catalyzes this reaction are therefore of importance in relation to the regulation of fatty acid synthesis. With the acetyl-CoA carboxylase purified from rat liver we obtained the following results.1. The active carboxyl in carboxylated acetyl-CoA carboxylase is bound, as in other carboxy-biofin-enzymes, to the l'-N-atom of biotin, which is amide-linked to the ~-amino group of a lysine residue in the enzyme protein.2. Enzyme activated by citrate at 25°C is reversibly inactivated by exposure to cold (0 to 4° C). Sucrose gradient centrifugafion studies in medium containing citrate indicate that the sedimentation coefficient of the enzyme protein is larger at 25°C than at 4°C.3. Enzyme is inhibited strongly by long-chain aeyl-CoA derivatives. The inhibition is competitive with regard to citrate and non-competitive with regard to aeetyl-CoA, bicarbonate or ATP. It was found by sucrose gradient centrifugation studies that the enzyme sediments more slowly in the presence of palmityl-CoA than in its absence.A possible significance of the in vitro findings in the cellular regulation of fatty acid synthesis is discussed. Inhibition of acetyl-CoA carboxylase by long-chain acyl-CoA derivatives might represent a negative feedback mechanism.
Article
Biotinamide and X-biotinyl derivatives of glycine, β-alanine, L-aspartic acid, L.-glutainic acid, L-lnicine, p-aminobenzoic acid and the diethyl esters of N-biotinyl-L-. asparLic and N-biotinyl-L-glutamic acids were examined microbiologically for biotin activity. Biotinamide, N-biotinyl-p-aminobenzoic acid and N-biotinyl-β-alanine have a spectrum of microbiological activity quite similar to that of biocytin, a form of biotin occurring in some natural products, but may be distinguished from biocytin by differences in solubility or acid stability. Representative compounds from the group synthesized readily combine with avidin.
Article
Alterations in the state of aggregation of liver chicken acetyl-CoA carboxylase in relation to its catalytic activity were investigated in the presences of various components participating in the carboxylation reaction. In the absence of citrate, ATP plus Mg2+, which inhibits the carboxylase, promotes dissociation of the enzyme. The inhibition as well as the dissociation by ATP plus Mg2+ is prevented by simultaneous addition of either acetyl-CoA or ADP. The effect of ADP is not observed in the presence of a large amount of bicarbonate.
Article
A modification of the naphthalene-dioxane-PPO liquid scintillator has been described which will allow up to 3.0 ml of an aqueous solution to be counted. The efficiency of this method in the presence of 1.0 ml water is 65.6% for C14 and 11.7% for tritiated water. This liquid scintillator has been used to count carbon-14 and tritium in urine, plasma, and liver homogenate. The effect of isotope concentration, solute concentration, and the presence of acids and bases on the count rate has been investigated.
Article
The effects of small, metabolically labile, intermediate “pools” on kinetics of isotope uptake and release are presented. The primary influence of such pools is to effect a coupling between components that are both derived and synthesized from the pool. Probably the most significant interaction is that of a component with itself. This is called “recycling”. It is shown that no matter how an isotope experiment is conducted, the true turnover rate or true half life cannot be calculated without information additional to the time course of activity in the component under study. It is noted that the presence of the pool does not alter the shape of kinetic curves. Therefore, the existence of such a pool cannot be adduced from kinetic experiments, and consequently the interpretation of the observed rate constant or the equivalent half life as either a “true” value or an “apparent” value is equivocal. Methods to evaluate the “true” turnover rates are considered.When two components are both connected to the same small pool, the turnover of one influences the other. The conditions determining whether this interaction is significant are discussed. Such coupling adds an additional term to, and changes the rate constants of, the original equation.If there are many components, there are many additional terms. It is shown that under certain conditions, the terms due to components other than the one under study may be combined in a way to give great simplification.Finally, the influence of growth on tracer kinetics is considered. A treatment employing specific activity data, yielding “the turnover rate constant for synthesis” is presented. This treatment is nearly correct for systems changing by a limited factor, say less than two fold. A procedure employing total activity measurement which yields the turnover rate constant for degradation is also presented.
Article
Earlier studies had shown that the enzyme tryptophan pyrrolase which cat lyzes the first irreversible step in the conversion of tryptophan to functional nicotinyl derivatives in rat liver, was strongly inhibited by NADPH which may be considered the ultimate distal product in the conversion of tryptophan to derivatives of nicotinamide. As an extension of this work the present study has demonstrated that the administration of nicotinamide in vivo to adrenalectomized animals resulted in a strong suppression of the tryptophan-mediated elevation of tryptophan pyrrolase but had little or no effect on the hormonal induction of this enzyme by corticosteroids. These effects of nicotinamide were in sofar specific as analogs of this compound had little or no effect on the specific tryptophan-medidated elevation of tryptophan pyrrolase. Furthermore, the effect of nicotinamide was shown to be specific for the enzyme, tryptophan pyrrolase, having no effect on the tryptophan-mediated increase in serine dehydratase. By means of anti-tryptophan pyrrolase antibodies prepared in rabbits, the pulse labeling of tryptophan pyrrolase in vivo by [14C]phenylalanine showed that, in animals pre-fed a 0% protein diet, tryptophan administration resulted in a doubling of the [14C]phenylalanine incorporation into tryptophan pyrrolase. Nicotinamide administration completely abolished this effect of tryptophan but had little effect on the dramatic stimulation of tryptophan pyrrolase synthesis by hydrocortisone. It is postulated that nicotinamide may exert its inhibitory effect on tryptophan induced synthesis of tryptophan pyrrolase at the level of genetic translation.
Article
Acetyl coenzyme A carboxylase has been isolated from chicken liver and purified more than 1000-fold in good yield. It is an unusually stable protein exhibiting negligible loss of enzymatic activity during 1 week of storage (1 to 5 mg of protein per ml) at room temperature in potassium phosphate buffer, pH 7. The pure enzyme catalyzes the carboxylation of 8.8 µmoles of acetyl-CoA (or 7.0 µmoles of propionyl-CoA) per min per mg of refractometrically determined protein at its pH optimum of 7.5 at 37°. The carboxylase-catalyzed carboxylation of acetyl-CoA is activated 15- to 16-fold by dl-isocitrate or citrate and 5-fold by malonate; tricarballylate is essentially inactive. Arrhenius plots for acetyl-CoA carboxylation in the presence or absence of isocitrate are biphasic, having a transition point at about 21°. Below this temperature, where there is very little activator effect, the temperature coefficient (Q10) in the presence of isocitrate is 8.0. Above the transition point, where isocitrate activation is evident, the temperature coefficient (Q10) in the presence of activator is 2.0 and in its absence is a negative value. At pH 7.5 and 37°, the carboxylase catalyzes the over-all reverse reaction (decarboxylation), ATP-32Pi exchange, malonyl-CoA-14C-acetyl-CoA exchange, and ADP-, Pi-, and Mg2+-independent malonyl-CoA decarboxylation reactions at 14%, 6.2%, 33%, and 1.2%, respectively, of the rate of the over-all forward (carboxylation) reaction. All of the above mentioned reactions exhibit essentially the same tri- and dicarboxylic acid activation patterns. Evidence is presented which indicates that isocitrate activation of the carboxylase is associated with an increased maximum velocity.
Article
1. The content of citrate in ;freeze-clamped' livers from starved and alloxan-diabetic rats was measured by using the specific citrate assay method of Gruber & Moellering (1966). 2. The content of citrate fell progressively during a period of 48hr. starvation to reach a plateau value that is 50% of the value for livers from fed rats. Some possible explanations for the conflicting reports of changes in hepatic citrate content during starvation are discussed. 3. The hepatic contents of ATP, pyruvate, lactate, glycogen and the hexose phosphates were decreased during starvation, whereas those of acetyl-CoA and AMP were increased. 4. Acute alloxan-diabetes produced similar changes in the contents of these metabolic intermediates. 5. The effects of starvation and diabetes on the citrate and acetyl-CoA contents are discussed in relation to control of gluconeogenesis, fatty acid synthesis and the activity of citrate synthase.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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