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THE UTILIZATION OF GLUCOSE 6-PHOSPHATE BY GLUCOKINASELESS AND WILD-TYPE

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... Whether intact nucleotides could enter the cell or whether they must be dephosphorylated before uptake was not known. Although most phosphorylated compounds are hydrolyzed before uptake, somesuch as L-a-glycerol phosphate (7), glucose 6-phosphate (5). glucose 1-phosphate (3), and other hexose phosphates (20)-are taken up in their intact form via active transport mechanisms. ...
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Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from [14C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [32P]AMP. Adenine inhibited uptake of [32P]AMP but not that of [32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of 32P and 14C from double-labeled AMP showed marked differences; 32P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide.
... The intake of glucose in E. coli involves two key enzymes: glucose kinase (glk) and protein-Npiphosphohistidine-D-glucose phosphotransferase (ptsG). Glucose kinase converts glucose to G-6-P with high catalytic efficiency [26][27][28]. Our laboratory has developed an E. coli strain, SG104, by deleting ptsG and enhancing glk to increase glucose intake [25]. ...
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Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. Conclusions: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.
... The striking difference between the growth of AG219 on the PTS sugar glucose and the non-PTS sugar glucose 6-phosphate (which is known to be taken up and utilized without dephosphorylation [9]) strongly supports the idea that a functioning PTS can lead to adequate pyruvate production in mutants devoid of pyruvate kinase activity. When glucosegrown cells of AG219 were assayed for glucose PTS activity by the method of Kornberg and Reeves (16), as expected, pyruvate production (19 nmol/min per dry weight of cells) occurred when glucose, but not when glucose 6-phosphate, was used in the assay. ...
Article
Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.
... Glucose-6-phosphate (G6P) can be actively transported by an inducible transport system in E. coli ML, K, and B without hydrolysis of the phosphate prior to its crossing the membrane (11,20,24). The effect of ghosts on the uptake of G6P in induced or uninduced E. coli B is shown in Fig. 7. ...
... Polyphosphate kinase (PPK) catalyzes the formation of ATP by transferring the terminal phosphate group of polyphosphate to ADP [35]. In the pentose phosphate pathways, hexokinase (HK) catalyzes the formation of glucose-6-phosphate using ATP [36], glucose-6-phosphate dehydrogenase (ZWF) catalyzes the formation of NADPH by oxidizing glucose-6-phosphate [37], phosphogluconolactonase (PGL) catalyzes the formation of D-gluconate 6-phosphate [38], and finaly 6-phosphogluconate dehydrogenase (GND) catalyzes the formation of NADPH and D-ribulose 5-phosphate [39]. The molecular weight of these enzymes and tRNA, polyphosphate, NADPH and ATP are all above 500 Da. ...
Article
5-Aminolevulinic acid (ALA) is an important cellular metabolic intermediate that has broad agricultural and medical applications. Previously, attempts have been made to synthesize ALA by multiple enzymes in cell free systems. Here we report the development of a semi-permeable system for ALA production using stable enzymes. Glucose, sodium polyphosphate, ATP, tRNA, glutamate and NADPH were used as substrates for ALA synthesis by a total of nine enzymes: adenylate kinase, polyphosphate kinase, glucose-6-phosphate dehydrogenase, phosphogluconolactonase, 6-phosphogluconate dehydrogenase, glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase from E. coli, hexokinase from yeast, as well as glutamyl-tRNA reductase and its stimulator protein glutamyl-tRNA reductase binding protein (GBP) from Arabidopsis in a semi-permeable system. After reaction for 48 h, the glutamate conversion reached about 95%. This semi-permeable system facilitated the reuse of enzymes, and was helpful for the separation and purification of the product. The ALA production could be further improved by process optimization and enzyme engineering. Abbreviations: PPK: polyphosphate kinase; ADK: adenylate kinase; ALA: 5-Aminolevulinic acid; HK: hexokinase; ZWF: glucose-6-phosphatedehydrogenase; PGL: phosphogluconolactonase; GND: 6-phosphogluconate dehydrogenase; GTS: glutamyl-tRNA synthetase; GTR: glutamyl-tRNA reductase; GBP: GTR binding protein; GSAAT: glutamate-1-semialdehyde aminotransferase.
... The intake of glucose in E. coli involves two key enzymes: glucose kinase (glk) and protein-Npiphosphohistidine-D-glucose phosphotransferase (ptsG). The glk converts glucose to G-6-P with high catalytic efficiency [26][27][28]. Our laboratory has developed an E. coli strain SG104 by deleting ptsG and enhancing glk to increase glucose intake [25]. ...
Preprint
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Background The biosynthesis of high value-added compounds through metabolically engineered strains has received widely attention in recent years. As an effective compound in pharmaceutical, cosmetic and food industry, myo-inositol (inositol) is mainly produced via a harsh set of chemical reactions from phytate. The proper distribution of carbon flux between cell growth and inositol production was a major challenge for constructing an efficient inositol-synthetic pathway. Recombinant E. coli strains have been constructed by metabolic engineering strategies to produce inositol, yet with a low yield. Therefore, construction of E. coli metabolically engineered strains with high stoichiometric yield will be attractive. Results In the present study, the recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained to efficiently synthesize inositol. Inositol was successfully biosynthesized after introducing two crucial enzymes, inositol-3-phosphate synthase (IPS) from Trypanosoma brucei , and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild type) and SG104 ( ΔptsG::glk , ΔgalR::zglf , ΔpoxB::acs ), a series of engineered strains for inositol production were constructed by deleting the key genes pgi, pfkA or pykF . Furthermore, the plasmid expression systems of IPS and IMP were optimized, and the gene zwf was regulated to enhance stoichiometric yield. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved with the combined strain R15 of SG104, Δpgi , Δpgm , and RBSL5-zwf. Simultaneously, the engineered strain R04 reached high-density fermentation level in a 1-L fermenter by using glucose and glycerol as mixed carbon source. In the scale-up bioconversion in situ with R04, 0.82 mol inositol/mol glucose was produced by fed-batch within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM). Conclusions The biosynthetic pathway of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provided an essential reference to obtain a suitable distribution of carbon flux between glycolysis pathway and product synthetic pathway.
... The intake of glucose in E. coli involves two key enzymes: glucose kinase (glk) and protein-Npiphosphohistidine-D-glucose phosphotransferase (ptsG). Glucose kinase converts glucose to G-6-P with high catalytic e ciency [26][27][28]. Our laboratory has developed an E. coli strain, SG104, by deleting ptsG and enhancing glk to increase glucose intake [25]. ...
Preprint
Full-text available
Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. Conclusions: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.
... The intake of glucose in E. coli involves two key enzymes: glucose kinase (glk) and protein-Npi-phosphohistidined-glucose phosphotransferase (ptsG). Glucose kinase converts glucose to G-6-P with high catalytic efficiency [26][27][28]. Our laboratory has developed an E. coli strain, SG104, by deleting ptsG and enhancing glk to increase glucose intake [25]. ...
Article
Full-text available
Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. Conclusions: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.
... Alternative glucose transporters, such as the low affinity galactose:H + symporter GalP and the ATP-dependent MglABC system, are able to internalize glucose in an unphosphorylated state, 27 while the ATP-dependent glucokinase (Glk) is able to phosphorylate glucose for glycolysis. 28 Several studies have further demonstrated that wildtype-like growth rates may be recovered in minimal medium supplemented with glucose in the absence of PTS when GalP is overexpressed. 29−31 In some strains, concomitant overexpression of Glk is required to fully restore growth. ...
Article
Tuning expression of competing endogenous pathways has been identified as an effective strategy in the optimization of heterologous production pathways. However, intervention at the first step of glycolysis, where no alternate routes of carbon utilization exist, remains unexplored. In this work we have engineered a viable E. coli host that decouples glucose transport and phosphorylation, enabling independent control of glucose flux to a heterologous pathway of interest through glucokinase (glk) expression. Using community sourced and curated promoters, glk expression was varied over a 3-fold range while maintaining cellular viability. The effects of glk expression on the productivity of a model glucose-consuming pathway were also studied. Through control of glycolytic flux we were able to explore a number of cellular phenotypes and vary the yield of our model pathway by up to 2-fold in a controllable manner.
... By corollary, GK and the mannose-PTS system provide the only mechanisms for glucose phosphorylation in S. lactis (Fig. 1). Similarly, GK and the two glucose-PTS systems (enzyme IIA-IIB and enzyme lIg1c-IlIg1c [19]) are the only physiologically significant mechanisms for glucose phosphorylation in E. coli (6,9,23). ...
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A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose-phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [14C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM. We conclude from our data that phosphorylation of glucose by S. lactis 133 can be mediated by only two mechanisms: (i) via ATP-dependent glucokinase, and (ii) by the phosphoenolpyruvate-dependent mannose-PTS system.
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Considerable differences in steady-state hexokinase specific activity were found in 16 N.C.I.B. strains of Klebsiella aerogenes grown in identical conditions in glucose-limited chemostats. Strains of N.C.I.B. 8258 had no detectable activity, but its glucose-phosphoenolpyruvate phosphotransferase specific activity and that of the other strains were closely similar, and it is concluded that this phosphotransferase activity regulates the overall utilization of glucose, in which hexokinase plays no essential role. The hexokinase activity was subject to regulation by the availability of phosphorus, but this did not affect the glucose phosphotransferase activity. tlactose-grown organisms (including strain N.C.I.B. 8258) had no glucose phosphotransferase activity, but more than adequate hexokinase activity to phosphorylate the intracellularly liberated glucose.
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Phosphofructokinase (pfkA) mutants of Escherichia coli are impaired in growth on all carbon sources entering glycolysis at or above the level of fructose 6-phosphate (nonpermissive carbon sources), but growth is particularly slow on sugars, such as glucose, which are normally transported and phosphorylated by the phosphoenolpyruvate, (PEP)-dependent phosphotransferase system (PTS).
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A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain beta-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by beta-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of omicron-nitrophenyl-beta-D-galactopyranoside (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H+/sugar cotransport. Entry of omicron-nitrophenyl-beta-D-galactopyranoside is less dependent on the need for proton reextrusion, probably because the stoichiometry of H+/substrate cotransport is greater for lactose than for ONPG.
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Several lines of evidence suggest that sucrose is transported by the lactose carrier of Escherichia coli. Entry of sucrose was monitored by an osmotic method which involves exposure of cells to a hyperosmotic solution of disaccharide (250 mM). Such cells shrink (optical density rises), and if the solute enters the cell, there is a return toward initial values (optical density falls). By this technique sucrose was found to enter cells at a rate approximately one third that of lactose. In addition, the entry of [14C]sucrose was followed by direct analysis of cell contents after separation of cells from the medium by centrifugation. Sucrose accumulated within the cell to a concentration 160% of that in the external medium. The addition of sucrose to an anaerobic suspension of cells resulted in a small alkalinization of the external medium. These data are consistent with the view that the lactose carrier can accumulate sucrose by a proton cotransport system. The carrier exhibits a very low affinity for the disaccharide (150 mM) but a moderately rapid Vmax.
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A systematic study of adenosine triphosphate (ATP)-dependent hexose kinases among microorganisms has been undertaken. Sixteen hexose kinases of five major types were partially purified from 12 microorganisms and characterized with respect to specificity for sugar and nucleotide substrates and Michaelis constants for the sugar substrates. Glucokinase activities that phosphorylate glucose and glucosamine are inhibited by N-acetyl-glucosamine and xylose, were found to be present in the non-sulphur photosynthetic bacteria Rhodospirillum rubrum, the blue-green algae Anacystis montana, and the protists Chlorella pyrenoidosa and Chlamydomonas reinhardtii (green algae), Hypochytrium catenoides (Hypochytridiomycete) and Saprolegnia Iitoralis (Oomycete). The myxobacteria Stigmatella aurantiaca contains a glucokinase activity with a different specificity pattern. Anacystis and Chlorella, besides their glucokinase activities, contain highly specific fructokinases, although that from Anacystis can also phosphorylate fructosamine; fructokinase from Anacystis has a molecular weight of 20 000, and exhibits a sigmoidal saturation curve for ATP when the Mg2+/ATP ratio is 2; this curve is transformed to a Michaelian one when under the same conditions an excess of Mg2+ (5 mM) is added. Saprolegnia however, besides the glucokinase, contains a mannofructokinase activity that phosphorylates mannose (Km 0.06 mM) and fructose (1 mM). On the other hand, hexokinase, a low specificity enzyme, was detected in the protist Allomyces arbuscula (Chytridiomycete) and in fungi Mucor hiemalis and Phycomyces blakesleeanus (Zygomycetes), and Schizophyllum commune (Basidiomycete). Schizophyllum contains a glucomannokinase activity together with hexokinase activity. The pattern of distribution of ATP-dependent hexose kinases among microorganisms seems to parallel that reported for biosynthetic pathways for lysine. The correlation with other biochemical parameters is also considered.
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The properties of an inducible hexose-phosphate transport system in Escherichia coli have been investigated. Mannose 6-phosphate, fructose 6-phosphate and glucose 6-phosphate are inducers as well as substrates of this transport system. A hexose phosphate transport-negative mutant was isolated which grew on the hexoses mannose, fructose, and glucose but not on the corresponding hexose 6-phosphates. This hexose-phosphate transport system is distinct from those for the corresponding hexoses as well as that for l-α-glycerophosphate. These strains of E. coli appear to be unable to transport galactose 6-phosphate and α-methylglucoside phosphate.
Article
During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.
Article
Genetic studies show that Escherichia coli has three enzymes capable of phosphorylating glucose: soluble adenosine 5'-triphosphate-dependent glucokinase, which plays only a minor role in glucose metabolism; an enzyme II, called glucosephosphotransferase, with high specificity for the D-glucose configuration; and another enzyme II, called mannosephosphotransferase, with broader specificity. The former enzyme II is active on glucose and methyl-alpha-glucopyranoside, whereas the latter is active on D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, and D-mannosamine. Mutations leading to loss of glucosephosphotransferase activity and designated by the symbol gpt are between the purB and pyrC markers in a locus previously called cat. The locus of mutations to loss of mannosephosphotransferase, mpt, is between the eda and fadD genes. Mutations to loss of glucokinase, glk, are between the ptsI and dsd genes.
Article
The bacterial phosphotransferase system participates in diverse physiological phenomena; its best characterized function is in the group translocation of sugars that are substrates of the system. Such sugars are phosphorylated as they are translocated across the cell membrane. Isolation of different proteins of the phosphotransferase system and reconstitution of the complex shows that in the net transfer of the phosphoryl group from phosphoenolpyruvate to a given sugar the phosphoryl group is sequentially transferred from one protein to another. In all cases so far studied, with one important exception, the phosphoryl group is linked to the proteins through a nitrogen atom in the imidazole ring of a histidyl residue. In the exceptional protein, the phosphoryl group is linked to a carboxy group. An additional function of the phosphotransferase system is to regulate the uptake of sugars that cannot be phosphorylated.
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In cultures of Escherichia coli W4597(K) and G34 under various nutritional conditions the rates of glucose utilization and cellular levels of fructose-1,6-P2 are quantitatively related by the Hill equation where the value of the Hill coefficient is approximately equal to 2. This is the first evidence that fructose-P2, or any metabolite which covaries with fructose-P2, modulates glucose utilization in E. coli. In light of previous observations from our laboratory this new observation and those in the succeeding report provide the first evidence that in E. coli glycolsis, glycogen synthesis and glucose utilization are coordinately regulated, thus providing for the coupling of ATP utilization and production under various metabolic circumstances. Alterations in the level of ATP apparently affect the velocity of phosphofructokinase, the rate-limiting enzyme in glycolsis, altering the cellular levels of glucose-6-P or fructose-P2. Changes in the levels of these hexose phosphates are quantitatively related to alterations in the rates of glucose utilization and glycogen synthesis in the intact E. coli cell.
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Uptake of 2-deoxyglucose, alpha-methylglucopyranoside, and glucose into intact cells of Brochothrix thermosphacta (formerly Microbacterium thermosphactum, ATCC 11509) was stimulated by KCN or CCCP. The glucose analogs were recovered almost totally as the sugar phosphates. Membrane vesicles were isolated from protoplasts and shown to be right side out by freeze fracturing and by using ATPase as a marker for the cytoplasmic membrane surface. Uptake of glucose into vesicles was dependent on the presence of phosphoenolpyruvate. NADH oxidation, K+ -diffusion gradients, and externally directed lactate gradients (pH greater than 7 initially) were used to generate transmembrane potentials across membrane vesicles. Above a threshold value of about -50 mV, uptake of glucose into membrane vesicles was reduced. Likewise, the maximum uptake of glucose and its two analogs into cells occurred when the protonmotive force was less than about -50 mV.
Article
Osmotic shock is a procedure in which Gram-negative bacteria are treated as follows. First they are suspended in 0.5 M sucrose containing ethylenediaminetetraacetate. After removal of the sucrose by centrifugation, the pellet of cells is rapidly dispersed in cold, very dilute, MgCl(2). This causes the selective release of a group of hydrolytic enzymes. In addition, there is selective release of certain binding proteins. So far, binding proteins for D-galactose, L-leucine, and inorganic sulfate have been discovered and purified. The binding proteins form a reversible complex with the substrate but catalyze no chemical change, and no enzymatic activities have been detected. Various lines of evidence suggest that the binding proteins may play a role in active transport: (a) osmotic shock causes a large drop in transport activity associated with the release of binding protein; (b) transport-negative mutants have been found which lack the corresponding binding protein; (c) the affinity constants for binding and transport are similar; and (d) repression of active transport of leucine was accompanied by loss of binding protein. The binding proteins and hydrolytic enzymes released by shock appear to be located in the cell envelope. Glucose 6-phosphate acts as an inducer for its own transport system when supplied exogenously, but not when generated endogenously from glucose.
Article
This chapter focuses on three devices: catabolite repression, transient repression, and catabolite inhibition, which regulate the utilization of many carbohydrates. Catabolite repression is a reduction in the rate of synthesis of certain enzymes, particularly those of degradative metabolism, in the presence of glucose or other readily metabolized carbon sources. Catabolite inhibition is a control exerted by glucose on enzyme activity rather than on enzyme formation, analogous to feedback inhibition in biosynthetic pathways. Catabolite repression influences many aspects of microbial growth and metabolism. In addition to the well known repressions of carbohydrate utilization and amino-acid degradation in bacteria and yeast, catabolite repression affects the formation of enzymes that function in the tricarboxylic acid cycle, glyoxylate cycle, fatty acid degradation, carbon dioxide fixation, and the respiratory chain. In higher organisms, catabolite repression has been observed in sugar cane, rats, and man. The question of whether catabolite repression acts to inhibit the transcription of DNA into m-RNA or to inhibit translation of messenger into protein has received conflicting answers. Catabolite repression is a control system that usually affects catabolic enzymes. If catabolite repression and transient repression are not mediated by the specific apo-repressor of each operon, there must be another protein that recognizes the low molecular-weight effector. The significance of a control mechanism, which influences the activity as opposed to the concentration of a carbohydrate-metabolizing enzyme is readily appreciated because bacteria have a limited ability to change enzyme concentrations.
Article
Both Li+ and Na+ stimulated the uptake of thiomethylgalactoside by the melibiose transport system ofEscherichia coli. On the other hand, Li+ inhibited the growth of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li+ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by -galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibiose transport. Li+ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of -galactosidase. Li+ was found to inhibit the entry ofp-nitrophenyl--d-galactoside, but notp-nitrophenyl--d-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies with different transport substrates.
Chapter
At first sight, bacteria appear to have a multiplicity of pathways for sugar degradation since a wide variety of five- and six-carbon sugars, sugar alcohols, sugar acids, and amino sugars can be utilized. For example, the commonly studied organism Escherichia colt can use more than 30 different monosaccharides for growth. However, the situation is more straightforward than it seems since the pathways for the catabolism of these compounds are interlinked and, with very few exceptions, they all produce glyceraldehyde-3-phosphate, which is converted by a trunk pathway (Fig. 1) to a common product of all sugar degradation, pyruvate.
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A constitutive stereospecific d-glucokinase was purified over 1000-fold from extracts of Aerobacter aerogenes PRL-R3. Only d-glucose (Km = 8 x 10⁻⁵m and d-glucosamine (Ki = 4 x 10⁻⁴m) were phosphorylated. The enzyme was inhibited by d-xylose (competitive with d-glucose, Ki = 3 x 10⁻³m) but not by 34 other sugars and related compounds tested. It was inhibited by adenosine diphosphate (competitive with adenosine triphosphate, Ki = 4 x 10⁻⁴m) but not by d-glucose 6-phosphate or d-mannose 6-phosphate. The pH optimum was 7.5 in glycylglycine buffer and about 8.9 in glycine buffer. Other properties studied were phosphoryl donor specificity, metal ion specificity, sedimentation coefficient, and stability. The product of d-glucose phosphorylation was identified as d-glucose 6-phosphate.
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A wild strain of Escherichia coli utilizes glucose 1-phosphate by hydrolysis of the ester at the cell surface, after which the glucose moiety but not the phosphate group enters the cell. However, when these cells are induced by treatment with glucose 6-phosphate, they are able to take up both glucose 6-phosphate and glucose 1-phosphate without preliminary hydrolysis. Mutant strains of E. coli that lack Enzyme I of the phosphotransferase system are unable to grow in the presence of glucose or glucose 1-phosphate as a carbon source. These strains show uptake of ¹⁴C-glucose 1-phosphate after being induced with glucose 6-phosphate. Mutant strains have been isolated that lack Enzyme I and are constitutive for the hexose phosphate transport system. They can grow on glucose 6-phosphate or glucose 1-phosphate, but not on glucose as carbon source. In these strains the uptake of ¹⁴C-glucose 1-phosphate involves entry of the entire molecule, and uptake is inhibited by glucose 6-phosphate. It is concluded that glucose 1-phosphate may be used by E. coli after preliminary hydrolysis by a surface phosphatase which is effective at neutral pH. In addition, glucose 1-phosphate is a substrate, but not an inducer, for the hexose phosphate transport system that is active with glucose 6-phosphate and related compounds.
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This paper presents evidence that the pathway by which d-fructose is metabolized in Aerobacter aerogenes PRL-R3 depends on whether it is presented to the cells extracellularly as the free hexose or intracellularly as a product of sucrose hydrolysis. Whereas the metabolism of exogenously supplied d-fructose is mediated by a phosphoenolpyruvate:d-fructose 1-phosphotransferase system and d-fructose 1-phosphate kinase (ATP:d-fructose 1-phosphate 6-phosphotransferase), the metabolism of the d-fructose moiety of sucrose is mediated by a specific d-fructokinase (ATP:d-fructose 6-phosphotransferase, EC 2.7.1.4) that is preferentially induced by sucrose. Conclusions drawn from studies with the wild type strain were supported by an analysis of mutants lacking d-fructokinase, d-fructose 1-phosphate kinase, or both of these enzymes. However, in contrast with the wild type, the mutant which lacks d-fructokinase metabolizes the d-fructose moiety of sucrose via d-fructose 1-phosphate rather than d-fructose 6-phosphate.
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An Escherichia coli mutant, DF2000, accumulates up to 0.06 m glucose 6-phosphate when supplied glucose. This extremely large concentration of intracellular glucose 6-phosphate does not serve to induce the active transport of this ester. However, if the medium is then supplemented with only 5 x 10⁻⁴m glucose 6-phosphate the transport system is induced. Thus, it appears that exogenous glucose 6-phosphate acts as an inducer but the endogenously formed ester is unable to do so. The ester accumulated by glucose-treated cells was isolated and purified. It proved to be an effective inducer of glucose 6-phosphate transport. The transport system for glucose 6-phosphate allows this ester to accumulate until the intracellular concentration is 20-fold greater than the concentration in the medium. When glucose-treated cells containing 0.06 m glucose 6-phosphate are induced for transport with 5 x 10⁻⁴m ester the initial concentration ratio is 120:1. Consequently, glucose 6-phosphate exits from the newly induced cells until a concentration ratio of 20:1 is established. Accumulation of glucose 6-phosphate by cells of the mutant given glucose results in growth stasis. This toxic effect is reduced or prevented by inducing levels of glucose 6-phosphate in the medium.
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2-Deoxy-d-glucose was taken up by Escherichia coli and was recovered from cell extracts largely as a material that behaved chromatographically as 2-deoxyglucose 6-phosphate. With ¹⁴C-labeled deoxyglucose, it was observed that radioactivity was taken up by the cell and partially released after 1 hour. No induction of transport for glucose 6-phosphate was observed on treatment of cells with 2-deoxyglucose, in spite of the apparent accumulation of intracellular 2-deoxyglucose 6-phosphate. By contrast, extracellular 2-deoxyglucose 6-phosphate, in low concentration, was an effective inducer of the hexose phosphate transport system. 2-Deoxyglucose inhibited growth of E. coli and also caused temporary growth stasis when glycerol or succinate was the carbon source. Cells growing in the presence of gluconate or pyruvate resisted these effects of 2-deoxyglucose. When glucose-grown cells were forced to adapt to glycerol or succinate as carbon source in the presence of 2-deoxyglucose, the lag period was prolonged for as long as several days. Often this was followed by rapid growth caused by spontaneous appearance of a mutant strain. The mutant was isolated and found to be resistant to growth inhibition by deoxyglucose.
Chapter
In reference to studies on the uptake of amino acids from blood, Waelsch(1) pointed out that “Experiments in which the amino acid concentration in blood is raised to an unphysiological level may elicit an aspect of the blood—brain barrier not operative under physiological conditions. For determining the uptake of amino acids of the brain under physiological conditions, accurate measurements of the arterio—venous differences would be required.” Although brain slices in vitro and other brain preparations have proved of considerable value in biochemical research, it is true that, as Geiger(2) observed, “they obviously do not possess all the metabolic machinery which is involved in the physiological activity of the nerve cell.”
Chapter
This chapter discusses the techniques that have been developed to isolate and characterize transport mutants, the criteria for identifying genes that affect transport directly, the linkage relationships of transport mutants in several organisms, the regulation of synthesis of transport systems, and the usefulness of mutants in understanding transport mechanisms. A variety of methods have been developed for the isolation of transport mutants, particularly in microorganisms. Some are selection methods, making use of conditions under which the transport mutant can grow but the wild-type cell cannot; others are merely screening methods, permitting the rapid identification of transport mutants among large numbers of wild-type cells. The particular strategy to be used in a given instance depends on the function of the transport system in question and on whether or not there are alternate routes for the substrate to enter the cell. The chapter tabulates basic information about the properties of existing transport mutants.
Article
The major objective of this chapter is to outline some of the important techniques which have been developed for the study of transport systems in bacteria. The techniques described are discussed in detail with respect to the study of the lactose transport system of Escherichia coli. It now appears that this transport system can serve as a useful model for a number of active transport systems in both bacterial and animal cells. In such transport systems, a protein (the “carrier”) embedded within the membrane mediates the translocation and accumulation of substrate; substrate appears within the cell without chemical modifications. The driving force for the accumulation of substrate is represented by the electrical and chemical forces acting on certain specific cations. In bacterial cells, accumulation by such transport systems is coupled to proton movements (see discussions by Mitchell, 1963, and Harold, 1972), whereas in animal cells such active transport is associated with the movement of sodium ions (for review, see Schultz and Curran, 1970). In the absence of energy coupling, however, these systems catalyze the facilitated diffusion of substrate across the cell membrane.
Article
The influence of different phosphate-compounds, especially of NaH2PO4×2 H2O and fructose-1,6-diphosphate, on nitrogen fixation was studied in three tribes ofAzotobacter chroococcum. In stagnant nonaerated cultures a phosphate-optimum exists for the nitrogenfixation of 90 µg P/ml. Using fructose-1,6-diphosphate the same quantity of nitrogen is fixed with only 50 µg P/ml. In aerated cultures the differences between inorganic phosphate and fructose-1,6-diphosphate (regarding their influence on the nitrogen-fixation) disappear. The quotient: used up C/fixed N also depends on aeration. In stagnant cultures containing organic phosphate-compounds, the C/N-quotient is lower than in those containing inorganic phosphate. In aerated cultures, however, the C/N-quotient is lowest with orthophosphate. The experiments indicate that the phosphate-esters are incorporated into the cell immediately without splitting up on the cell surface. These incorporated phosphateesters in non-aerated cultures, lacking an extensive oxidative phosphorylation, are apparently important for the economy of nitrogen fixation. The ecological role of the incorporation of phosphate-esters is discussed in relation to the fact that a great part of phosphorus in the natural environment is not inorganic but organic phosphate.
Article
This chapter discusses the identification and reconstitution of anion exchange mechanisms in bacteria. Work with vesicles and proteoliposomes shows that the chemiosmotic transport of sugar phosphates occurs, not by proton-symport, but by anion exchange, in both gram-positive and gram-negative bacteria. Further analysis shows the existence of a variable stoichiometry that allows homologous sugar phosphate exchange to mediate an unexpected net uptake of substrate. Because this exchange causes the influx of protons along with the sugar phosphate, continued transport depends on maintenance of a pH gradient (alkaline inside), thereby ensuring that the Pi-linked exchangers take part in the overall chemiosmotic circuitry of the cell. The study of Pi-linked exchange has defined not only their physiological and biochemical properties, but has also been the vehicle for development of a particularly simple set of protocols designed to study such proteins in an artificial liposomal system. Such methods that take advantage of the stabilizing effects of osmolytes (e.g., glycerol), have made it possible to use reconstitution in an analytical fashion, not just as a preparative tool.
Article
Dank einer Lysetechnik mit Penicillin kann man in einer Bakterienpopulation eine Heterogenität gegenüber einem in die Membran induzierbaren Markierer wie zum Beispiel ein Transportsystem, feststellen. Diese Methode wurde verwendet, um zu zeigen, dass nach einer vollständigen Induktion einer Permease in einer Population eine Heterogenität nach drei Generationen auftritt. Diese Heterogenität ist der Existenz von Wachstumszonen in der Membran zuzuschreiben, die mit den Ruhezonen abwechseln, so dass die ursprüngliche Membran in eine begrenzte Anzahl von Gebieten zerstückelt wird, die einer gegrenzten Anzahl von Nachkommen vererbt werden, vier im Falle unserer Bakterienkultur. In dem vorliegenden Artikel wird diese Methode angewandt, um die Aufspaltung einer grossen Anzahl von in die Membran induzierbaren Markierern, den bekannten Permeasen, einer Phosphotransferase (des Enzymes II des D-Mannitols) einer Oxydoreduktase, der anaeroben Nitrat-Reduktase, und einigen unbekannten Markierern, wahrscheinlich Transportsystemen, die durch das Glukonat bzw. die Trehalose induziert werden, zu entdecken.
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A mutant of Escherichia coli unable to metabolize succinate has been used to study the nature of succinate transport in whole cells. It is demonstrated that cells accumulate succinate against a concentration gradient. The uptake of succinate is competitively inhibited by fumarate and malate. The Michaelis constants for the dicarboxylic acids are all in the range of 15 to 30 µm. The accumulation of succinate is prevented by various energy poisons and sulfhydryl reagents. Inhibitors of mitochondrial dicarboxylate transport are without any effect on the succinate uptake system of E. coli. The influx and efflux of succinate in whole cells is temperature dependent. The influx increases rapidly between 15 and 36° and efflux between 36–50°. The transport system is induced by succinate and repressed by glucose. Mutants lacking adenyl cyclase and some lacking enzyme I of phosphotransferase system acquire succinate transport activity when grown in the presence of succinate and cyclic 3′,5′-AMP. Mutants of E. coli unable to transport succinate fall into two broad categories which are referred to as dct (dicarboxylate transport) and ct (carboxylate transport). The dct mutants fail to grow on dicarboxylic acids (fumarate, malate, and succinate) but grow normally on the monocarboxylic acid, lactate. The ct mutants do not grow on either the dicarboxylic acids or on lactate. Both classes of mutants grow well on acetate. The dct class of mutants can be genotypically separated into two categories. The dct A mutants map at around 69 min, and the dct B mutant maps at about 17 min (linked to galactose locus) on the genetic map. The ct mutants are also linked to galactose. It is inferred from these experiments that there are at least two components involved in the transport of succinate in E. coli. It is suggested that the ct gene possibly specifies a carboxylate binding protein. This suggestion is based on the evidence that osmotically shocked cells of E.coli lose the capacity for uptake of succinate.
Article
Galactoside permease of Escherichia coli (E. coli) is one of the most studied transport systems in bacteria. There are various reasons for many experiments being devoted to this transport system. Some of the reasons include—(1) the clear genetic analysis of the structural gene, controlling the synthesis of P-galactoside permease, has helped focus on the specificity of transport systems and has provided a methodology of general applicability, and (2) the major methodological advantage of P-galactoside permease is the current availability of nonmetabolizable substrates, the thiogalactosides, of which the best examples are methyl-β-D-thiogalactoside (TMG), β-D-galactosyl-β-D-thiogalactoside (TDG), and isopropyl-β-D-thiogalactoside (IPTG). The kinetics of uphill transport of thiogalactosides is discussed in the chapter. The uptake of radioactively labeled thiogalactosides can be studied in fairly physiological conditions by the fast Millipore filtration technique. The time course of such an uptake is represented diagrammatically. Uphill transport is thermodynamically active and its energy requirement has been established by several independent methods. One of these is the inhibition of uphill transport by uncoupling agents such as sodium azide or dinitrophenol.
Article
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Mutants of E. coli limited in their ability to synthesize phosphatidic acid become severely restricted in their capacities for growth and macromolecular synthesis. The paper reports on the effects of partial limitation of phosphatidic acid synthesis in a mutant of E. coli, resulting from an altered sn glycerol 3 phosphate acyltransferase (EC 2.3.1.15). The alteration in the K(m) of this enzyme for sn glycerol 3 phosphate was correlated with intracellular glycerol P concentration, rates of lipid and macromolecule synthesis, and growth rate. The reduction in the rate of lipid synthesis correlated well with the reduction in macromolecule synthesis and growth rate and suggested a rapid mechanism for coordination of these processes. Inhibition of lipid synthesis caused a specific reduction in the size of the ATP (adenosine triphosphate) pool and it is postulated that an activation of ATPase by altered membrane environment may be the possible mechanism of ATP reduction and subsequent loss of synthetic capacities.
Article
A mutant strain of Escherichia coli K12 grows well on galactose, lactose, or arabinose, but exhibits poor growth on glucose and a number of other common carbon sources. Defective growth on glucose is partially overcome by prior growth on galactose. Resting cells and growing cultures utilize galactose in preference to glucose when both sugars are present in the medium, although the enzymes for glucose utilization are present in normal amounts. However, the repressive effect of glucose on inducible enzyme formation is lacking. The mutant is characterized by a marked lack of crypticity toward β-galactosides, and lactose is rapidly taken up and hydrolyzed by resting cells. Uptake of galactosides is not inhibited by glucose, contrary to what is observed in the wild type. The defect appears to be in the transport mechanism for glucose. This defect results in a lack of control by glucose of galactose or galactoside uptake.
Article
An enzyme, phosphoramidic hexose transphosphorylase, which catalyzes a phosphoryl transfer from monophosphoramidate, N-phosphorylgylcine or monophosphoryl histidine to glucose has been purified about 160-fold from extracts of succinate-grown Escherichia coli. Several aldo- and ketohexoses as well as sedoheptulose have been shown to be phosphoryl acceptors with this enzyme. Certain preparations of phosphoramidic hexose transphosphorylase have been shown to produce only glucose 1-phosphate while others produce both glucose 1-phosphate and glucose 6-phosphate, suggesting the presence in the latter fractions of a second phosphoryl transferring system.
Article
Both acid and alkaline phosphatase have been demonstrated in strains of E. coli. Bacterial acid phosphatase (pH optimum 4–5) displays a fairly high specificity for the hexosephosphates, while the alkaline enzyme (pH optimum 8.5–9.5) hydrolyzes all the phosphomonoesters tested.The kinetics of formation of both enzymes have been studied and it has been shown that alkaline phosphatase in measurable amount is only formed when Pi becomes limiting in the medium, at which point the enzyme is formed in substantial amount at a maximum rate.The implications of the findings with respect to mechanisms of controlling enzyme formation are discussed.
Article
There was no significant change in flow rates of parotid saliva in nineteen of twenty subjects while they viewed photographs of lemons, or in fourteen of twenty subjects while they cut lemons in a glove box. Neither parotid nor whole-mouth secretion changed from baseline when subjects viewed fresh lemons and lemonade presented in a plastic box. Further, no significant changes in whole-mouth secretion rates were observed when subjects viewed photographs of two appetizing foods, or of fresh doughnuts in a plastic box, even though subjects knew they could eat the doughnuts after the experiment. In most cases, sniffing of the lemons or of the doughnuts resulted in increased flow rates. Subjects demonstrated large differences in their patterns of affective responses to full-strength and diluted lemon juice, which were independent of salivary flow. In the absence of olfactory or tactile stimulations, few subjects altered parotid or whole-mouth secretion rates in response to viewing food or photographs of food. A reevaluation of findings on 'psychic' stimulation of saliva may be in order to ascertain the role of olfactory, tactile, and even trigeminal clues in salivary response to food stimuli.
Article
Glucose oxidase (EC 1.1.3.4) is the most widly employed enzyme as analytical reagent. This is the result of (1) its utility in the determination of glucose, an analyte of wide analytical interest, and (2) its relatively low cost and good stability that make the glucose/glucose oxidase system a very convenient model for method development (particularly in the area of biosensors). This review discusses in detail enzyme structure, biocatalysis, enzymes as analytical reagents, properties of glucose oxidase (including a historical account), and the use of glucose oxidase as an analytical reagent in homogeneous systems as well as an immobilized reagent.
Article
The experiments reported in this paper show that osmotically active spheres prepared from E. coli respond osmotically to the addition of galactosides. The characteristic of this response are similar to those of galactoside-permease: inducibility, inhibition by azide, specificity. Furthermore, the increase in internal osmotic pressure manifested by the change in optical density, is of the same order as would be calculated from the total amount of galactoside known to be accumulated by whole cells.It is concluded that the bulk of the intracellular galactoside accumulated by the action of galactoside-permease is a free solute. If any intracellular binding of galactosides occurs, it is quantitatively insignificant. The extension of this conclusion to other permease-like systems is a matter for speculation.The method described is capable of detecting a change in the internal osmotic pressure equivalent to 0.01 M sucrose and probably somewhat less.
Article
The properties of the glucose-transport system of Escherichia coli K10 were studied by following intracellular accumulation of 14C-labeled α-methylglucoside. A mutant defective in the uptake of glucose was found to be unable to accumulate α-methylglucoside to a significant level, confirming the view that the carrier for glucose also acts on α-methylglucoside. Although the glucoside was not incorporated into cellular constituents or metabolized for energy, about one-half of the accumulated molecules underwent phosphorylation while one-half remained as the free glucoside. Studies on the inhibition of uptake of the glucoside showed that the affinity of the compound depended upon the substituents on C-2, C-3 and C-6. Structural modifications at C-1, on the other hand, had relatively little effect on the affinity unless a large aglycone was added. α-Methylglucoside accumulated by the cells was gradually lost upon dilution of the cells in simple inorganic medium. The rate of exit could be accelerated by two groups of compounds: non-metabolizable structural analogs with affinity for the transport carrier and metabolizable compounds which entered the cell by other routes. The effect of the latter group depended upon their further metabolism within the cell rather than their own direct effects on the carrier system.
Rickenberg's laboratory at Indiana University Similar observations with other strains have been made at Vanderbilt University by B. M. Pogell who obtained evidence for an inducible glucose 6-phosphate permease (personal communication)
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Kessler in H. Rickenberg's laboratory at Indiana University. Similar observations with other strains have been made at Vanderbilt University by B. M. Pogell who obtained evidence for an inducible glucose 6-phosphate permease (personal communication). 22 Schmidt, G., and S. J. Thannhauser, J. Biol. Chem., 161, 83 (1945).
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