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B Shane

Publications and source records attributed to B Shane.

At least 91 records · Page 5Linked to original sources
PubMed1981

Folylpoly-gamma-glutamate synthesis by bacteria and mammalian cells.

The purification and properties of folylpolyglutamate synthetase from Corynebacterium sp, and some properties of partially purified enzyme from Lactobacillus casei, Streptococcus faecalis, Neurospora crassa, pig liver, and Chinese hamster ovary cells, are described. The Corynebacterium enzyme catalyzes a MgATP-dependent addition of glutamate to a variety of reduced pteroate and pteroylmono-, di-, and triglutamate substrates, with the concomitant production of MgADP and phosphate. Although glutamate moieties are added in a sequential fashion, the kinetic mechanism, which is Ordered Ter Ter, precludes the sequential addition of glutamate moieties to enzyme-bound folate. It is suggested that catalysis precedes via the formation of a pteroyl-gamma-glutamyl phosphate intermediate. The in vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differ from source to source, appear to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources. Only one enzyme appears to be involved in the conversion of pteroylmonoglutamates to polyglutamate forms in both bacteria and mammalian cells. Bacterial folylpolyglutamate synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydro-pteroylpolyglutamates as their preferred, and sometimes only, polyglutamate substrate. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of mammalian folylpolyglutamate synthetases.

Adenosine Triphosphate↗
PubMed1981

Radiometric--microbiologic assay of vitamin B-6: application to food analysis.

A radiometric microbiologic assay for vitamin B-6 was applied to food analysis. The method was shown to be specific, reproducible and simpler than the standard turbidimetric microbiologic technique. The analysis of seven commercially available breakfast cereals was compared to a high performance liquid chromatography method. Three out of the seven cereals agreed when assayed with both methods (P greater than 0.1). Four cereals, however, differed in value considerably (P less than 0.05). Further studies are required to determine whether these differences were due to different extraction procedures used. The study showed that the new radiometric-microbiologic method can be used to measure total vitamin B-6 or, combined with a column separation procedure, to analyze for specific forms of the vitamin.

Biological Assay↗
PubMed1980

Pteroylpoly(gamma-glutamate) synthesis by Corynebacterium species. Purification and properties of folypoly(gamma-glutamate) synthetase.

Folylpolyglutamate synthetase was purified 7000-fold from extracts of Corynebacterium sp. The final preparation, which was greater than 95% pure, had a monomer molecular weight of 53,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to a variety of reduced pteroate and reduced pteroylmono-, di-, and triglutamate substrates with the concomitant production of ADP and Pi. Although the specificity for the folate substrate was wide, the pteroate and pteroylmonoglutamate substrates were utilized much more effectively than the polyglutamate derivatives. The most effective substrates were tetrahydropteroate, tetrahydrofolate, and 5,10-methylene-tretrahydrofolate. The most effective diglutamate substrate was 5,10-methylene-tetrahydropteroyldiglutamate. Addition of more than one glutamate moiety was only observed with tetrahydropteroate and 5,10-methylene-tetrahydropteroylmono- and diglutamates as substrates. The enzyme exhibited a preference for ATP as the nucleotide substrate. dATP was almost as effective while UTP and CTP were less effective substitutes. The specificity for L-glutamate appeared to be absolute. Enzyme activity was maximal at about pH 10 and exhibited an absolute requirement for a monovalent cation, of which K+ was the most effective. Preliminary studies suggest that the active form of the enzyme may be a dimer and that K+ may be required to effect dimerization.

Adenosine Triphosphate↗
PubMed1980

Pteroylpoly(gamma-glutamate) synthesis by Corynebacterium species. Studies on the mechanism of folypoly(gamma-glutamate) synthetase.

The mechanism of action of folylpolyglutamate synthetase from Corynebacterium sp. was investigated using tetrahydrofolate, MgATP, and glutamate as the substrates. Initial velocity, product inhibition, and competitive inhibition studies were consistent with an Ordered Ter Ter mechanism with MgATP binding first to the enzyme, tetrahydrofolate second, and glutamate last. The order of dissociation from the enzyme was ADP, folate, and Pi. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. The Michaelis constants for (dl)-tetrahydrofolate, MgATP, and glutamate were 2,1 muM, 18 MUM, and 160 muM, respectively. Beta, gamma-Methylene-ATP was a very effective inhibitor of the reaction with an affinity for the enzyme 1 order of magnitude greater than that of ATP.

Adenosine Diphosphate↗
PubMed1980

Pteroylpoly(gamma-glutamate) synthesis by Corynebacterium species. In vivo synthesis of folates.

Corynebacterium sp. synthesizes pteroylpolyglutamates which are excreted into its growth medium. The major intracellular folates were identified as 10-formyltetrahydro-, 5-formyltetrahydro-, tetrahydro-, and 10-formylpteroyltetraglutamates. The major folates in the medium were identified as 10-formylpteroyltri- and tetraglutamates. p-Aminobenzoic was metabolized to these compounds without any appreciable buildup of pteroylmono- or diglutamate derivatives. The rate-limiting steps in the elongation of the glutamate chain of folate derivatives appear to be at the level of formation of a pteroate derivative and at the pteroyltetraglutamate level.

Biological Transport↗
PubMed1980

Folate assay: a comparison of radioassay and microbiological methods.

Folate mono- and polyglutamate standards were assayed by a microbiological method and by four commercially available radioassay methods to evaluate the usefulness of radioassay techniques for the quantitation of naturally occurring folates. Folate monoglutamates exhibited different responses in the radioassay procedures, depending on the one-carbon constituent and oxidation state. Folate polyglutamates exhibited an increased response that varied depending on the folate concentration. The varied responses of folate mono- and polyglutamates in the radioassay procedures make this technique unsuitable for the determination of the mixture of folate derivatives that are normally encountered in biological extracts.

Biological Assay↗
PubMed1978

Absorption of mono- and polyglutamyl folates in zinc-depleted man.

The effect of experimental zinc depletion on the intestinal absorption of mono- and polyglutamyl folates was assessed in six healthy male subjects by measuring the increase in serum folate after an oral dose of pteroylmonoglutamate and pteroylheptaglutamate before and after initiation of a zinc-deficient diet. At the beginning of the study, no differences were detected in the absorption of pteroylmono- and heptaglutamate. After zinc depletion, the rise in serum folate after an oral dose of pteroylheptaglutamate was reduced by an average of 53% while pteroylmonoglutamate absorption appeared to be unchanged. These data suggest that zinc depletion results in a decreased intestinal hydrolysis of pteroylpolyglutamate and that intestinal conjugase in man is a zinc-dependent enzyme.

Adult↗
PubMed1978

Availability of monoglutamyl and polyglutamyl folates in normal subjects and in patients with coeliac sprue.

Intestinal folate absorption was assessed in six normal subjects and in four patients with coeliac sprue who were studied before and after treatment by dietary gluten exclusion. Comparisons were made of the luminal disappearance from the perfused jejunum of 3H-pteroylmonoglutamate and pteroyl 14C-glutamylhexaglutamate, and of the 48-hour urinary recovery of each isotope after perfusion and a tissue saturating dose of folic acid. The labelled urinary folates consisted of folic acid, 10-formyltetrahydrofolate, and 5-methyltetrahydrofolate. In each group urinary recovery of 3H was significantly greater than that of 14C, confirming the evidence from jejunal perfusion that the availability of monoglutamyl folate is greater than that of polyglutamyl folate. According to the urinary recovery data, both folates were poorly absorbed in untreated coeliac sprue, but were normally absorbed after treatment. Assuming uniform displacement of the absorbed labelled folates by the parenteral flushing dose, the finding of greater urinary isotope recovery than of luminal folate disappearance from the perfused proximal jejunal segment suggests an adaptation of the distal small bowel for folate absorption in coeliac sprue.

Adult↗
PubMed1977

Identification of glutamate chain lengths of endogenous folylpoly-gamma-glutamates in rat tissues.

A simplified procedure for the determination of the glutamate chain lengths of endogenous tissue folate is described. Natural pteroylpoly-gamma-glutamates in tissue extracts, irrespective of their one-carbon moiety, were converted by a reductive cleavage procedure to a homologous series of p-aminobenzoylpoly-gamma-glutamates, differing only in glutamate chain length. Desalting and concentration of the extracts were achieved by absorbing the derivatives on active charcoal followed by their elution with ethanol:ammonia. Aminobenzoylpolyglutamates were separated by DEAD-cellulose chromatography and quantitated by a colorimetric procedure for primary aromatic amines. The major endogenous folates in rat liver and kidney were pteroylpentaglutamate derivatives with smaller amounts of pteroyltetra- and hexaglutamate.

Animals↗
PubMed1977

Uptake and metabolism of [3H]folate by normal and by vitamin B-12- and methionine-deficient rats.

The uptake of an injected dose of [3H]folic acid and its metabolism to pteroylpoly-gamma-glutamate forms by the livers and kidneys of vitamin B-12- and methionine-deficient and -supplemented rats were investigated. The initial hepatic uptake of the labeled folate dose was the same in deficient and supplemented animals, demonstrating no involvement of vitamin B-12 or methionine in folate transport. At longer time periods, a decreased hepatic net uptake of labeled folate was observed in the deficient animals compared to supplemented animals, and this was directly correlated with the decreased ability of the deficient animals to synthesize pteroylpolyglutamates. The absolute rate of loss of labeled pteroylmonoglutamate from liver was the same in deficient and supplemented animals. These data are best explained by a modification of the 'methyl trap' hypothesis for the interrelationship of vitamin B-12 and folate metabolism. Vitamin B-12 deficiency can lead to lowered levels of 5-methyltetrahydrofolate:homocysteine methyltransferase, creating a functional folate deficiency by 'trapping' an increased proportion of folate as the methyl derivative. In addition, as methyltetrahydrofolate is a poor substrate for folylpoly-gamma-glutamate synthetase, there is a decreased synthesis of pteroylpolyglutamates, the forms of the vitamin that are preferentially retained by tissues. This results in decreased tissue folate levels under conditions of vitamin B-12 deficiency. Vitamin B-12 and methionine deficiency had no significant effect on the distribution of endogenous pteroylpolyglutamates in rat liver and kidney, although total endogenous folate in rat liver was reduced by about 60%. The distribution of labeled pteroylpolyglutamates in rat liver and kidney 48 h after the tracer dose of [3H]folate closely resembled the endogenous distribution in these tissues.

Animals↗
PubMed1977

Metabolism of 5-methyltetrahydrofolate by Lactobacillus casei.

The metabolism of 5-[Me-14 C]methyltetrahydrofolate in Lactobacillus casei proceeded oxidatively with incorporation of label into purine and thymidylate derivatives. No labelled methionine was formed. (l)-5-Methyltetrahydrofolate, the natural isomer, was not a substrate for the L. casei folylpoly-gamma-glutamate synthetase although the unnatural (d)-isomer was slowly metabolized to the diglutamate form.

Glutamates↗
PubMed1977

Rate-limiting steps in folate metabolism by Lactobacillus casei.

Oxidation of 5-methyltetrahydrofolate to 5,10-methylenetetrahydrofolate was the rate-limiting step in 5-methyltetrahydrofolate metabolism by Lactobacillus casei. The limiting steps in the utilization of suboptimal levels of folate by L. casei were related to the ability of folates to function in purine and/or thymidylate biosynthesis. Folates with glutamate chains of up to at least seven residues were substrates for these biosynthetic enzymes, and comparisons of bacterial growth yields with transport rates for these folates indicated that the polyglutamates were more effective substrates in purine and thymidylate synthesis than the corresponding pteroylmonoglutamates. Lactobacillus casei contained low levels of a B12-independent, pteroylpolyglutamate-specific methionine synthetase. Its methylenetetrahydrofolate reductase also functioned more effectively with pteroylpolyglutamate substrates.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1976

Transport and utilization of methyltetrahydrofolates by Lactobacillus casei.

Transport of labeled 5-methyltetrahydrofolate (5-methyl-H4PteGlu) and 5-methyl-H4PteGlu5 by Lactobacillus casei was via a carrier-mediated system. Both the natural (l) and unnatural (d) diastereoisomers were transported. Active transport was only demonstrated with the monoglutamate. Comparisons of growth rates with transport rates demonstrated that pteroypolyglutamates were more effectively utilized by L. casei than the monglutamate derivatives. The rate-limiting step in the utilization of higher polyglutamates was their transport into the cell, and in the case of the oxidized polyglutamates, their reduction. Utilization of monoglutamates was not limited by their transport but by metabolism, which also explains the different types of growth response curves observed between monoglutamates and higher polyglutamates. This phenomenon, sometimes called positive drift, prevents normalization of these growth curves. The rate-limiting step with the monoglutamates appears to be either the metabolism of 5-methyl-H4PteGlu or their conversion to polyglutamate forms.

Biological Transport, Active↗
PubMed1976

Transport and metabolism of vitamin B6 in the yeast Saccharomyces carlsbergensis 4228.

Active transport of pyridoxine, pyridoxal, and pyridoxamine occurs in resting cells of Saccharomyces carlsbergensis 4228 and can lead to intracellular concentrations of free vitamin much higher than those supplied externally. The initial Km for pyridoxine uptake is 3.6 x 10(-7) M at 30 degrees and pH 4.5, which are optimum for growth. Transport is inhibited by many unphosphorylated vitamin analogs, the most effective being 5'-deoxypyridoxine, 5'-deoxypridoxal, toxopyrimidine, 4'-deoxypyridoxine, and 3-amino-3-deoxypyridoxine. Two distinct uptake systems that differ in structural specificity and ionic requirements are present. One, with optimum pH of 3.5, transports pyridoxal effectively, but not pyridoxamine; the other (optimum pH 6.0) transports pyridoxamine effectively, but not pyridoxal. Both systems transport pyridoxine, while neither transports pyridoxal 5'-phosphate. Other properties of these systems are similar, indicating that they share certain elements in common. An initial temperature optimum of 30 degrees is observed for pyrodoxine transport and, at this temperature, an "overshoot" in intracellular vitamin levels, with subsequent decrease to a constant level, occurs with time. It appears that intracellular vitamin, or a derivative, activates the exit mechanism for the vitamin. Exit rates also depend on the resuspension buffer and are increased in the presence of glucose and decreased by azide. Above 30 degrees net uptake of pyridoxine drops initially, then rapidly increases to a second optimum at 50 degrees; the uptake system is inactivated at about 55 degrees. The optimum at 50 degrees apparently results from activation of inflow as exit is rapid and is accelerated by azide. No overshoot was detected at 50 degrees, so it appears that the exit system is not regulated by intracellular vitamin at this temperature. A phase transition in membrane lipids occurs at 30 degrees and may be responsible for the change in properties of the inflow and exit mechanisms above this temperature.

Binding, Competitive↗
PubMed1975

Transport and metabolism of folates by bacteria.

Transport of labeled folic acid (PteGlu), pteroylpolyglutamates (PteGlu3-5), 5-methyl-tetrahydrofolate (5-methyl-H4PteGlu), and methotrexate in late-log phase cells of Lactobacillus casei was active, and subject to inhibition by unlabeled pteroylmonoglutamates, pteroylpolyglutamates, and iodoacetate, but not glutamate or glutamate dipeptides. Pteroylpolyglutamates were transported without prior hydrolysis and shared a common uptake system with pteroylmonoglutamates. The affinity and maximum velocity of PteGlun uptake decreased with increasing glutamate chin length (Km:PteGlu1, 0.03 mum; PteGlu3, 0.32 mum; PteGlu4, 1.9 mum; PteGlu5, 3.7 mum) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by L. casei, once transported, than monoglutamate. No concentration of 5-methyl-H4PteGlu3-8 inside the cells was observed. The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 mum) were 10-formyl-H4PteGlu2 and 10-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No 10-formyl-Pte-Glu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 nm) metabolized the vitamin to polyglutamate forms, the major derivatives being H4PteGlun. First order exit rates of labeled folate from preloaded L. casei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 min-1 to greater than 0.002 min-1 as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n larger than or equal to 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released. Pediococcus cerevisiae actively transported 5-methyl-H4PteGlu but did not take up to 5-methyl-H4PTeGlu3-8. No active accumulation of 5-methyl-H4PteGlu was observed in Streptococcus faecalis.

Azides↗