1.1. A procedure of isolation of non-histone proteins from rat liver chromatin in mild conditions provided 3 groups of these proteins, i.e. NHCP1, NHCP2 and NHCP3.2.2. The investigated proteins are devoid of DNA and revealed various influences on RNA synthesis in vitro.3.3. The extraction of rat liver chromatin with 0.35 M NaCl (pH 7.5) removed about 30% of examined proteins. Electrophoretic patterns of 3 groups of non-histone proteins from total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins are compared.
1. The fraction of proteins extracted from nuclei with 0.35 M NaCl and soluble in 2% trichloroacetic acid was examined in five Morris hepatomas and rat liver. 2. This fraction was a much greater percentage of the total 0.35 M NaCl soluble proteins in the tumors than in normal or regenerating liver. 3. In part, this difference was due to proteins with molecular weights greater than high mobility group proteins. 4. The conditions for precipitation of high mobility group proteins 1 and 2 with trichloroacetic acid were found to differ in hepatoma and liver fractions.
Previous studies have suggested that human lactoferrin has a bacteriostatic effect on Escherichia coli 0111 growth. Determination of the time required for cultures to reach one-half maximal cell density (t1/2) indicates that within the concentration range of lactoferrin used in this study, its effect in vitro on the growth of E. coli 0111 is kinetic rather than bacteriostatic. Compared to a control, added apo-lactoferrin (250-1000 micrograms/ml) produced only a delay effect as seen by an increase in the t1/2 indicating these concentrations are probably within the subinhibitory concentration range. The kinetic delay effect of apo-lactoferrin is also consistently increased in the presence of Zn2+ and Cu2+ cations. Cu2+, Zn2+ and NTA (nitrilotriacetate) did not affect the growth rate of this organism in the absence of lactoferrin compared to the control. These studies indicate that the mechanism by which lactoferrin alters the bacterial growth of E. coli 0111 is more complex than simple iron deprivation.
1.1. γ-Glutamyltranspeptidase and a peptidase which cleaves cysteinylglycine and its S-derivatives are localized within the brush border membrane of the proximal straight tubule.2.2. The transpeptidase has previously been shown to be an amphipathic protein in which the membrane binding and catalytic domains are separated by a papain-sensitive sequence of amino acids.3.3. The Triton-purified peptidase exhibits a higher apparent subunit molecular weight (150,000) than the papain-purified peptidase (135,000).4.4. The presence of the papain-sensitive sequence of amino acids was required for the incorporation of the Triton-purified peptidase into lecithin vesicles.5.5. Papain treatment of isolated brush border membrane vesicles results in 90–95% solubilization of transpeptidase and peptidase activities without altering the internal volume of the vesicles.6.6. At least 92% of both activities associated with vesicles can be inactivated with specific Fab antibodies or precipitated by indirect immunoprecipitation.7.7. The non-accessible γ-glutamyltranspeptidase and peptidase activities were shown to be associated with the internal surface of a unique population of inverted vesicles.8.8. Therefore, the two activities are orientated exclusively on the outside of the brush border membrane, consistent with their proposed participation in the extracellular degradation of glutathione and mercapturic acid precursors.
1. Cationic fractions were isolated from a low chromium (less than 0.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system. 2. Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides. 3. Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast. 4. Additional activity was present in the yeast GTF sample, which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified. 5. The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question. 6. The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast systems as an assay for GTF.
1. Neocuproine binding to ceruloplasmin markedly increases the chlorpromazine-ceruloplasmin-catalyzed oxidation of NADH. 2. 1,10-Phenanthroline and 2,2'-dipyridyl inhibit neocuproine activation in a competitive manner. 3. The order of enzyme chelator complex stability was: phenanthroline greater than dipyridyl greater than neocuproine.
1. Reaction of 1,2-cyclohexanedione with arginine residues of egg white riboflavin-binding protein results in a loss of the binding activity. 2. In borate buffer pH 8.0, with 0.15 M cyclohexanedione, the inactivation proceeds with a pseudo-first-order rate constant 0.084 hr.-1. 3. At least 65% of lost riboflavin binding capacity can be recovered on 12 hr incubation in 0.5 M hydroxylamine pH 7.0. 4. All 5 arginine residues are modified, 2-3 of them seem to react much easier than others. 5. The correlation between modification of arginines and protein inactivation, as analyzed by kinetic and statistical methods, suggests that one of low-reactivity residues is "essential" for riboflavin binding. 6. In the holoprotein, one arginine residue is almost completely protected from 1,2-cyclohexanedione modification. 7. Riboflavin does not dissociate from holoprotein, even on prolongated incubation with the reagent. 8. The protected arginine residue seems to be located in the riboflavin binding pocket of protein macromolecule.
1. The effect of chronic alcohol consumption, catalase inhibitor 3-amino-1,2,4-triazole (amino-triazole) and peroxisome proliferator clofibrate on the level of Fe/ADP-ascorbate-induced lipid peroxidation has been studied in the rat myocardium. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. Combined us well as separate treatment with ethanol (36% of dietary calories) and aminotriazole caused elevation of the rate of lipid peroxidation in the nuclear-free homogenate or total particulate fraction of the rat heart. The most pronounced effect was noted during combined application of ethanol and aminotriazole. 3. Prolonged clofibrate treatment significantly increased the level of nonenzymatic lipid peroxidation in the rat myocardium. 4. Peroxidative alteration of the myocardial lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). Separate ethanol, aminotriazole or clofibrate treatment did not affect the level of u.v. absorption of lipids from the total particulate fraction. However, when ethanol and aminotriazole were administered simultaneously an increase of conjugated diene formation was observed. 5. The data obtained confirm the hypothesis that ethanol or clofibrate-induced activation of the myocardial lipid peroxidation may be due to the increase of hydrogen peroxide-generating capacity of the heart microperoxisomes.
1. We have used 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to investigate autoregulation of homologous receptor and the control of c-myc mRNA and protein expression in C3H/10T1/2 cells. 2. 10 nM 1,25-(OH)2D3 stimulated 1,25-(OH)2D3 receptor (VDR) synthesis in both non-transformed C3H/10T1/2 Cl 8 and in chemically transformed C3H/10T1/2 Cl 16 cells within 4 hr of treatment. Maximal induction was observed between 8 and 24 hr. 3. Two VDR mRNA transcripts, 2.7 and 4.8 kb, were present in both cell types. There were parallel changes in VDR specific mRNA levels and cellular VDR concentration in the C3H/10T1/2 Cl 8 cells indicating that the increase in receptor concentrations was dependent on de novo mRNA synthesis. 4. The increase in VDR mRNA concentration in the chemically transformed C3H/10T1/2 Cl 16 cells was maximal already at 4 hr, preceding the maximal increase in receptor concentration by 4-6 hr. 5. Analysis of c-myc mRNA levels also showed cell line specificity. 6. The c-myc mRNA level increased 2.1-fold with 10 nM 1,25-(OH)2D3 treatment in C3H/10T1/2 Cl 8 cells after 12 hr while the C3H/10T1/2 Cl 16 cells had maximal c-myc mRNA level after 1 hr. 7. The relative amount of c-myc mRNA remained higher than that of unstimulated controls the next 10-12 hr in C3H/10T1/2 Cl 16 cells. 8. The c-myc protein levels were not affected by 1,25-(OH)2D3 treatment in either cell line as detected by Western blot analysis. 9. Our data suggest that 1,25-(OH)2D3 mediated induction of VDR does not require prior c-myc protein synthesis in the C3H/10T1/2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
1. An endoxylanase (EC 188.8.131.52) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile "Caldocellum saccharolyticum" strain Tp8T184.108.40.206. It was found to have an M(r) of 42,000 and an isoelectric point of approx. 5.0. 2. The enzyme showed optimum activity at pH 5.0-7.7 and had an activation energy of 44 kJ mol-1. It was stable at room temperature at pH 4.5-11.5 in the presence of 0.5 mg ml-1 bovine serum albumin. The half-life of the enzyme at 75 degrees C was 20 min at pH 6.0 in the presence of 0.5 mg ml-1 bovine serum albumin. 3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The Km for oat spelts xylan was 0.021% (w/v) at pH 6.0. 4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl beta-D-cellobioside and p-nitrophenyl beta-D-cellobioside, but could not hydrolyse xylobiose. 5. It showed transferase activity on p-nitrophenyl beta-D-xylopyranoside. Xylose did not inhibit the enzyme.
1. The activation of S-adenosyl-L-methionine decarboxylase (SAM-DC) by putrescine and a series of 1,4-butanediamines with a substituent in position 2 was studied. 2. Kinetic data show the activation of SAM-DC by putrescine is essentially uncompetitive. 3. All 2-substituted 1,4-butanediamines were activators, although not as potent as putrescine itself. 4. At high concentrations of SAM activation of SAM-DC by putrescine and putrescine analogs deviated considerably from uncompetitive activation kinetics. 5. In order to explain the experimental data, especially the non-linearity of the "fractional velocity plots", it was necessary to postulate two independent, but equivalent activator binding sites, for which substrate (SAM) and activator compete. 6. Based on this kinetic model an equation was derived which describes the rate of SAM decarboxylation as a function of substrate and activator concentrations. 7. From the simulated curves, approximate values for equilibrium constants for the binding of activator and substrate to the activator binding sites, and relative rate constants for the product forming steps were calculated. 8. Even a minor change of the structure, such as the substitution of one hydrogen atom by fluorine in the 2-position of putrescine had a very considerable effect on the potency of activation. 9. It is apparent that the structural requirements of an activator of SAM-DC are highly specific.
1.1. The syntheses of N,N′-o-phenylenedi[1,4-14C]maleimide (60–70% yield) and the unlabeled ortho and para isomers (40–50% yield) have been carried out on a milligram scale.2.2. Use of the 14C-labeled compound with Escherichia coli succinic thiokinase showed that the crosslinking agent combined with the β subunit first in the formation of αβ dimer. Incorporation of about 2.5 moles of the reagent (40% of this bound bifunctionally) was sufficient to inhibit enzyme activity completely.
1.1. The porphyrogenic agent, 3,5-diethoxycarbonyl-l,4-dihydrocollidine (DDC) produces a marked inhibition of hepatic ferrochelatase activity when injected in vivo. It has no effect in vitro.2.2. Following the administration of DDC to mice or rats, an inhibitor of ferrochelatase activity has been isolated from livers and resolved and purified by LH-20 column chromatography. Mice which are more sensitive to DDC produce more inhibitor substance than rats and control livers possess small but measurable quantities of the inhibitor substance.3.3. The inhibitor has porphyrin-like properties with characteristic visible and fluorescence spectra and can be separated from protoporphyrin IX. When [14C]aminolevulinic acid was injected 2 hr prior to DDC, the purified inhibitor substance contained high radioactivity.4.4. Cycloheximide pretreatment of animals did not affect the amount of inhibitor formed, although this treatment is known to markedly reduce δ-aminolevulinic acid synthetase activity and the porphyria produced by DDC.5.5. These results indicate that the formation of an inhibitor after the administration of DDC is due to the catabolism of hepatic heme to a substance with porphyrin-like properties and with profound inhibitory characteristics toward ferrochelatase activity.
1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.
Mitochondrial and soluble Type I and Type II hexokinase from various rat tissues differed in their susceptibility to inhibition by glucose-1,6-bisphosphate (Glc-1,6-P2). In tissues where Type I is the predominant form, the mitochondrial enzyme was less susceptible to inhibition by Glc-1,6-P2 than the soluble enzyme, especially at high Mg2+ concentration. In tissues where Type II is the predominant form, the mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 than the soluble enzyme, especially at low Mg2+ concentration. The results suggest that changes in the intracellular concentrations of Glc-1,6-P2 and Mg2+ under various conditions would affect the activity of the bound and soluble hexokinase from different tissues in a different manner.
The following parameters were determined in the rabbit psoas muscle after perfusion in the presence of either insulin, propranolol, or isoproterenol: Concentrations of cyclic AMP, glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose-1-phosphate, glucose 6-phosphate, and fructose-1,6-bisphosphate. Maximum and "regulatory" activities of the enzymes glycogen phosphorylase, glycogen synthase, phosphofructokinase, and histone-phosphorylating protein kinase.
1.1. Human and rat liver Fructose 1,6-bisphosphatase have low specific activities at physiological pHs.2.2. Cysteine, threonine, serine and glycine activate the enzyme present in cytosols. A small amount (5%) of the enzyme is bound to microsomes. This fraction does not require any activator for maximal activity.3.3. A factor that is capable of activating the cytosol enzyme has been isolated and partially purified from liver microsomes and co-elutes with the microsomal-bound enzyme. A similar factor has been isolated from the cytosol fraction of 2 hepatomas.
1.1. Human fructose 1,6-bisphosphatase (EC 220.127.116.11) is composed of subunits of 44,000 daltons ±9% and exists predominantly as a dimer in 0.005 M Tris-glycine buffer, pH 18.104.22.168. The human enzyme is more sensitive to 5′-AMP inhibition (I (0.5) 3.2 × 10−5 M) than that of 2 other ominivorous species, i.e. the albino rat (2.3 × 10−4 M) and the Syrian hamster (3.6 × 10−4 M).3.3. The circular dichroism spectrum reveals highly oriented thyrosine and phenylalanine residues and an alpha helix content of 23%.4.4. As all the phosphatases previously studied, the enzyme has a low specific activity at physiological pHs.
1. Among eleven tissues of rat, the liver type of fructose 1,6-bisphosphatase (FBPase) subunit was detected in the liver, kidney, testis, pancreas and lung by Western blot analysis using anti-(liver FBPase) or anti-(muscle FBPase) serum. 2. The muscle type of the enzyme subunit was detected only in the pancreas other than skeletal muscle. Both types of the enzyme subunit were found in the pancreas. 3. Neither anti-(liver FBPase) nor anti-(muscle FBPase) serum detected the band of enzyme subunit on the blots of the extracts of brain, heart, small intestinal mucosa, spleen and placenta. 4. FBPase is present in fetal rat liver at least as early as the 14th day of gestation. 5. In agreement with the increase in immunological staining density, the level of the enzyme activity in fetal liver increased exponentially during fetal development. 6. The muscle enzyme was not detected until the fetus reached the 19th day of gestation.
1.1. For the determination of relationship between FDP and ATP in the rat liver pyruvate kinase regulation, kinelic studies have been carried out at several ATP and FDP concentrations.2.2. The results obtained on FDP activation show a great cooperativity for FDP saturation with a Hill coefficient of h = 22.214.171.124. Kinetic studies on ATP inhibition also show a great cooperativity for ATP saturation (h = 2.84) at high FDP concentrations.4.4. These results may contribute to explain the regulation of rat liver pyruvate kinase accounting for the activity of this enzyme at high FDP concentrations modulated by small changes in ATP concentrations.
1. The level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was found to be strikingly decreased in brains of adult rats (5 months of age) as compared to young (10-14 days of age). 2. This age-related decrease in Glc-1,6-P2, the potent inhibitor of hexokinase and activator of phosphoglucomutase, was accompanied by a correlated increase in the activity of hexokinase and a reduction in phosphoglucomutase. 3. Evidence is provided showing that Glc-1,6-P2 participates in the regulation of these enzymes' activities with age. 4. The age-related changes in Glc-1,6-P2 and in the enzymes' activities in brain were opposite to those which we previously found in skeletal muscle. 5. These results suggest that Glc-1,6-P2 is involved in the regulation of carbohydrate metabolism during growth in both brain and muscle, as well as in the interrelationship between these two tissues.
1.1. Phospholipase A induced an activation of glucose 1,6-diphosphate (Glc-1,6-P2) phosphatase (the enzyme which degradates Glc-1,6-P2), and decreased the levels of Glc-1,6-P2 in the isolated rat diaphragm muscle.2.2. The fall in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a concomitant reduction in these enzymes' activities.3.3. Lysolecithin, the product of phospholipase A action on membrane phospholipids, mimicked all these effects of phospholipase A.4.4. Phosphofructokinase and phosphoglucomutase from phospholipase A or lysolecithin-treated diaphragms, were extremely sensitive to activation by Glc-1,6-P2.5.5. These results suggest that a close relationship exists between phospholipase A action on membrane phospholipids, and the regulation of glucose metabolism in muscle mediated by Glc-1,6-P2.
1. Dietary excess histidine caused an increase in the total activity of fructose 1,6-bisphosphatase, and a decrease in 6-phosphofructokinase in the liver. 2. The hepatic concentrations of free histidine and lysine were higher in rats fed a histidine-excess diet. 3. The addition of histidine, lysine or arginine to the assay mixture for fructose 1,6-bisphosphatase resulted in a significant increase in its activity. The 6-phosphofructokinase activity in the liver was not enhanced by the addition of histidine to the assay mixture.
1. The effects of physiologic concentrations of insulin on the contents of glucose 1,6-bisphosphate (glucose 1,6-P2) and regulators of glucose 1,6-P2 synthase in intact human skeletal muscle have been investigated. 2. Insulin increased glucose 1,6-P2 from a basal value of 70 +/- 6 to 135 +/- 12 mumol/kg dry wt (P less than 0.001). 3. Activation of synthase could not be associated with changes in its inhibitors (fructose 1,6-P2, Pi, citrate) or its substrate glucose 6-P.
A metabolic system in vitro, which converts fructose 1,6-biphosphate into the two alternative products, lactate and glycerol phosphate, was designed to study the distribution of metabolic fluxes and, specifically, the control of glycerol phosphate production rate in rat muscle extract. Experiments were carried out at several protein concentrations by dilution of rat muscle extract, showing non-linear behaviours of flux versus protein concentration. These were hyperbolic for glycerol phosphate and logarithmic for L-lactate. The influence of the flux towards any alternate product on the rate giving the other was studied by stimulation of each. Results obtained show that in this system, flux towards glycerol phosphate is not affected by lactate production and the same occurs for the contrary case. Glycerol phosphate dehydrogenase seems to be the only enzyme in this system whose activity controls the flux towards glycerol phosphate.
1. Injection of epinephrine induced in skin a decrease in the level of glucose-1,6-bisphosphate (Glc-1,6-P2), which was accompanied by correlated changes in the activities of several enzymes which are modulated by this regulator. 2. These effects were blocked by the alpha adrenergic blocker phentolamine, in contrast to muscle where the hormone increases Glc-1,6-P2, acting through beta receptors. 3. The changes in the enzymes' activities, as well as in glycogen and lactate content induced by epinephrine, reveal that the hormone causes, in skin, a stimulation of glycogenolysis and glycolysis, as well as an acceleration of pentose phosphate pathway. 4. The reduction in glycogen content induced by epinephrine, was blocked by the beta adrenergic blocker propranolol, whereas the hormone's effects on the other processes were mainly mediated through alpha receptors.
1.1. Glucose-1,6-diphosphate (Glc-l,6-P2), the powerful regulator of glucose metabolism, was found to be strikingly decreased in muscle of old rats.2.2. This decrease may result from the increase in the activity of Glc-1,6-P2 phosphatase, the enzyme which degradates Glc-1,6-P2.3.3. The fall in Glc-1,6-P2 in old age was accompanied by a concomitant reduction in phosphoglucomutase activity. However, phosphofructokinase activity was elevated, most probably as a result of the decrease in ATP and increase in ADP and AMP.4.4. These results indicate that in the course of aging glycolytic capacity of muscle is not reduced and the alterations reflect a more anaerobic metabolism.5.5. We also found a marked rise in plasma creatine phosphokinase in old animals, which may point to membrane abnormalities.
1. Fructose 1,6-bisphosphatase was assayed in crude extracts of physiologically important organs and tissues in the ostrich. 2. Highest activity was found in liver and lowest in brain tissue. 3. No activity was detected in the heart, gizzard or adrenals. 4. The enzyme was purified in homogeneous, apparently undegraded form from liver utilizing Blue dextran-Sepharose affinity chromatography. 5. The enzyme is similar to mammalian fructose 1,6-bisphosphatase in many respects including its indispensability of Mg2+ for catalytic activity. 6. Relative molecular weight of the native enzyme and its subunit is about 150,000 and 35,000 respectively. 7. The amino acid composition of ostrich liver fructose 1,6-bisphosphatase is distinctly different from that of the chicken muscle enzyme, but compares favourably with the composition of the rabbit liver enzyme. 8. The purified enzyme is devoid of tryptophan.
Injection of serotonin (5-hydroxytryptamine) induced a marked decrease in the level of glucose 1,6-diphosphate (Glc-1,6-P2) in the rat tibialis anterior muscle. Concomitant to the decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, the activities of both these enzymes were markedly reduced by serotonin. The level of Glc-1,6-P2 and the activities of phosphofructokinase and phosphoglucomutase increased with age in the tibialis anterior muscle and the effect of serotonin was more pronounced in the older animals. Serotonin also induced a significant increase in the level of cyclic GMP in muscle. The serotonin-induced changes in the normal muscle mimic the changes in carbohydrate metabolism we found previously in muscular dystrophy.
1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg2+ and Mn2+. 2. Mg2+ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory. 3. Mn2+ produces a 10-fold rise in Vmax higher than Mg2+. [Mn2+]0.5 is much larger than [Mg2+]0.5. At elevated [Mn2+] inhibition is observed. 4. Mg2+ and Mn2+ produce antagonistic effects on the inhibition of the enzyme by high substrate. 5. Fru-2,6-P2 inhibits the enzyme by rising the S0.5 and favouring a sigmoidal kinetics. 6. The inhibition by Fru-2,6-P2 is released by Mg2+ and more powerfully by Mn2+ increasing the I0.5.
1. The extent of liver injury assessed as elevation of plasma transaminases was decreased 40-50% by administration of fructose 1,6-diphosphate to rats receiving the highly hepatotoxic combination of chlordecone and CCl4. 2. This protection was accompanied by significantly higher sustenance of ATP levels in the liver. 3. Polyamine synthesis as well as interconversion were stimulated in favor of maintaining higher levels of polyamines. 4. These events are consistent with the concept that suppressed hepatocellular regeneration which leads to progression of otherwise limited injury observed in chlordecone potentiation of CCl4 hepatotoxicity is due to lack of cellular energy.
The intracellular concentration of glucose-1,6-bisphosphate (Glc-1,6-P2) in rat tibialis anterior muscle was markedly decreased following the injection of bradykinin. Injection of bradykinin also induced a significant increase in the level of cyclic GMP in muscle. The activity of glucose-1,6-bisphosphatase, the enzyme that degrades Glc-1,6-P2, was markedly enhanced by bradykinin, which may account for the decrease in the level of Glc-1,6-P2. The decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a concomitant reduction in these enzymes' activities. The bradykinin-induced decrease in Glc-1,6-P2 and in the activity of phosphofructokinase, the rate-limiting enzyme in glycolysis, may be involved in the pathogenic influences of this hormone in various clinical conditions.
Injection of trifluoperazine abolished the bradykinin-induced decrease in intracellular concentration of glucose 1,6-bisphosphate (Glc-1,6-P2) in rat tibialis anterior muscle and skin. These changes in Glc-1,6-P2 levels may be attributed to the changes in the activity of glucose 1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2), which was markedly enhanced by bradykinin and reversed by trifluoperazine. Concomitantly to the changes in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, the activities of these enzymes were reduced by bradykinin and restored by trifluoperazine. These findings suggest that trifluoperazine treatment may have a beneficial effect on the depressed glycolysis induced by bradykinin in tissue damage.
In this minireview the properties and characteristics of plant fructose-1,6-bisphosphatases (D-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 126.96.36.199) are discussed. The properties and characteristics of the chloroplastic and cytoplasmic forms of the enzyme are reviewed. For purposes of comparison some reference is made to fructose-1,6-bisphosphatases from other species.
1.1. Local anesthetics, lidocaine and procaine, which were reported to cause muscle damage, induced a marked decrease in the level of glucose-l,6-diphosphate (Glc-l.6-P2) in the isolated rat diaphragm muscle.2.2. Concomitant to the decrease in Glc-l.6-P2, the powerful activator of phosphofructokinase and phosphoglucomutase, the activities of these enzymes were significantly reduced.3.3. Both local anesthetics also exerted a decrease in the concentration of muscle ATP, the effect of lidocaine being more pronounced than that of procaine.4.4. All these changes induced by the local anesthetics closely resembled dystrophic muscle, suggesting a common mechanism associated with the ultrastructural damage of muscle in both conditions.
1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations. 2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor. 3. Mg2+ decreases the inhibition produced by AMP and ADP by enhancing their I0.5 and completely annulates the inhibitory effect of ATP. 4. In the presence of Mn2+ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.
1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.
The enzyme D-glycero D-ido octulose 1,8-bisphosphate:D-altro-heptulose 7-phosphotransferase (abbreviated to phosphotransferase, PT) catalyses the transfer of the phosphate ester group at C-1 between altro-heptulose (sedoheptulose) and octulose phosphate intermediates of the L-type pentose pathway. Using synthetically prepared and 14C-labelled octulose mono- and bisphosphates, two methods are described for the measurement of the catalytic capacity of the PT reaction operating in both the "forward" and "reverse" modes of L-type pentose pathway operation. PT activity was found in normal, regenerating and foetal rat liver, rat heart, rat epididymal fat pad, rat kidney, brain and skeletal muscle, extracts of C. fusca, pea leaf and a variety of tumour tissues. The highest activity of the enzyme was found in the neoplasms. The Michaelian kinetic constants, temperature and pH optima for the reaction of the enzyme from rat liver together with an assortment of its substrate specificities have been determined. Vanadate anion was found to inhibit the enzyme and the pattern of inhibition suggests that the PT may act by a sequential mechanism. Neither arabinose 5-phosphate nor inorganic phosphate showed any effect on the catalytic activity of the PT enzyme in liver.
1.1. Temperature dependent kinetic studies of the activation of Pyruvate oxidase (EC 188.8.131.52), in the presence of cofactors and sodium dodecyl sulphate, indicate that the enzyme exists in different conformations at 0 and 25°C.2.2. In addition these two conformation have differential sensitivities to Urea. 3. Activation by sodium dodecyl sulphate also affects the pH stability profile for the enzyme.3.4. The sensitivity of the oxidase to N-ethylmaleimide indicates the presence of a sensitive sulphydryl group which can be protected from modification by the preincubation with cofactors Mg2+ and thiamine pyrophosphate.
1.1. Total low density lipoprotein (TLDL) spectra of plasma samples from humans with type III hyperlipoproteinaemia and plasma from rabbits which had been fed a diet containing added cholesterol for 0 to 43 days were determined by analytical ultracentrifugation.2.2. The comparison showed very similar TLDL spectra between the two groups when the spectra were taken individually or averaged.3.3. Variations in the response of rabbits to dietary cholesterol are reported and discussed.
1. Parathyroid hormone-induced down-regulation was studied in the osteosarcoma cell line UMR-106. 2. A maximal priming does of bPTH (1-84) down-regulated PTH-responsiveness to 40% of its initial value; bPTH (1-41) was less effective than bPTH (1-84), whereas bPTH (42-84) had no effect, alone or in combination with bPTH (1-41). 3. A tentative model for the function of different domains of parathyroid hormone in down-regulation is suggested.
12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC, EC 184.108.40.206) in normal, preneoplastic and malignant rat brain cells in culture, but treatment with phorbol, acetate or medium shift resulted in a similar response. Medium shift induced ODC activity in C3H/10T1/2 CL8 cells 4 and 12 hr after treatment. TPA induced only the 12 hr peak. ODC induction in C3H/10T1/2 CL8 cells was completely inhibited by cycloheximide and actinomycin D. Addition of alpha-amanitin abolished the 12 hr peak, but the TPA induced ODC activity was only partly inhibited. ODC induction by TPA was lower in C3H/10T1/2 CL8 cells initiated with 3-methyl-cholanthrene (MCA). ODC increased with TPA up to 10(-7) M and decreased at higher concentrations of TPA.
1. Nuclear fraction of cortex and synaptosomal fraction of hippocampus of rat brain were preincubated with ATP and different, "second messengers" as well as some "classical" neurotransmitters and then incubated with Glp6[125I]Tyr8SP6-11 (JP). 2. It was found that different neurotransmitters and especially "second messengers" might be involved in the activation or inhibition of peptidase I and II. 3. Activation of different protein kinases results in substantial modulation of degradation of JP (SP C-terminal fragment). 4. The data confirm that products of one peptidase might be inhibitors of the second peptidase acting on JP, although the second enzyme might be affected additionally in conditions activating different protein kinase systems.
1. Peptidase(s) activity of nuclear and synaptosomal fraction from cortex and hippocampus of rat brain against pyroGlu6[125I-Tyr8]SP6-11 was evaluated in different concentration of Ca2+, Mg2+, K+ and Na+ in about "isotonic" conditions. 2. The effects of studied ions on the peptidase activities forming N-terminal and C-terminal fragments are different especially in synaptosomes of both areas. 3. The differences of ionic requirements for N- and C-forming activities are particularly relevant for Ca2+ at the cortex and K+ at the hippocampus. 4. Ca2+ activate forming of N-terminal fragments in the nuclear fraction whereas inhibit it in synaptosomes from both areas. 5. The ionic requirements for C-terminal fragments' formation in synaptosomes of both areas are contradictory.
The purification and properties of an enzyme from Nocardia sp. which catalyses the conversion of p-hydroxybenzonitrile to p-hydroxybenzoic acid and ammonia without intermediate formation of the amide is described. The enzyme displayed a broad pH optimum between 7.0 and 9.5 and exhibited Michaelis-Menten kinetics with Km of 1.27 mM for p-hydroxybenzonitrile. The 12-unit multimeric enzyme possessed a mol. wt of 560,000 and was sensitive to thiol-specific reagents. Although aliphatic nitriles were not substrates for the enzyme a broad range of substituted aromatic nitriles were attacked with a general preference being shown for those with meta substitution.
1. In sheep alpha alpha 113His and alpha D alpha alpha 113His globin gene haplotypes, the percentage gene efficiencies, from the 5' to the 3' end, are about 32-18 and 30-14-6, respectively. 2. We previously found that in a alpha alpha alpha alpha homozygote there may be a 1-2% alpha 113His chains, which however were difficult to resolve from alpha 113Leu chains by CMC chromatography. 3. We report here the experimental conditions which allowed a neat resolution of the 1% alpha 113His chain peak by RP-HPLC. 4. This finding strongly suggests the occurrence of the alpha D alpha alpha alpha 113His haplotype where the percentage gene efficiencies are 30-(14-5)-1, supporting the existence of a gradient of decreasing expression in multiple alpha-globin gene haplotypes.