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B Shane

Publications and source records attributed to B Shane.

At least 73 records · Page 4Linked to original sources
PubMed1987

Mammalian folylpoly-gamma-glutamate synthetase. 3. Specificity for folate analogues.

A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folylpolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the gamma-carboxyl of the terminal glutamate in the correct position for catalysis. Steric limitations imposed on the internal glutamate residues that loop out and additional steric constraints imposed by binding of different pterin moieties would be expected to effect slight conformational changes in the protein and/or the terminal glutamate and would explain the decrease in on rate and catalytic rate with increased polyglutamate chain length, and the differential effect of one-carbon substitution on the catalytic rate with polyglutamate derivatives. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Mammalian folylpoly-gamma-glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis.

The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B12 status, can be mimicked in vitro by using purified enzyme. Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B12 deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly. 4-Aminofolates are metabolized to retainable forms at a slow rate compared to folates. Although folate accumulation by cells is not very responsive to changes in folylpolyglutamate synthetase levels and cellular glutamate concentrations, cellular accumulation of anti-folate agents would be highly responsive to any factor that changes the expression of folylpolyglutamate synthetase activity.

Animals↗
PubMed1986

In situ autoradiographic detection of folylpolyglutamate synthetase activity.

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.

Animals↗
PubMed1986

The Reitan-Indiana Aphasia Screening Test: scoring and factor analysis.

This article presents a scoring system and factor analysis for the Reitan-Indiana Aphasia Screening Test. Eight content categories were clinically identified and scored (N = 197). Also, the interrelationships of these content areas were explored by factor analysis. The analysis revealed two major factors, a general language abilities factor and a sensorimotor coordination factor. The pattern of loadings was interpreted with reference to hypothesized relationships and previous research on the test. The findings also have implications for the clinical interpretation of test results.

Brain Diseases↗
PubMed1986

Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells.

Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folic acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.

Animals↗
PubMed1985

Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product.

The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.

Cloning, Molecular↗
PubMed1985

Vitamin B12-folate interrelationships.

The studies discussed in this review support the view that biochemical and clinical symptoms common to both folate and vitamin B12 deficiency are due to the induction of a functional folate deficiency, which in turn is induced by cobalamin deprivation. The interrelationship between these two vitamins is best explained by the methyl trap hypothesis stating that vitamin B12 deficiency can lead to lowered levels of methionine synthetase, which results in a functional folate deficiency by trapping an increased proportion of folate as the 5-methyl derivative. In addition, as 5-methyl-H4PteGlu is a poor substrate for folylpolyglutamate synthetase, there is a decreased synthesis of folylpolyglutamates and consequently a decreased retention of folates by tissues. The real folate deficiency that ensues because of decreased tissue folate levels is probably as important physiologically as the functional deficiency caused by the methyl trap. The sparing effect of methionine can be explained by adenosylmethionine inhibition of methylenetetrahydrofolate reductase, which would prevent the buildup of 5-methyl-H4PteGlun. A deficiency in vitamin B12 would not, in itself, be sufficient to cause a disturbance in folate metabolism. The deficiency would have to result in lowered methyltransferase levels before any such disturbance would be manifest.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1983

Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase.

Folylpolyglutamate synthetase was purified 200,000-fold from extracts of Lactobacillus casei. The homogeneous protein was a monomer of Mr = 43,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to 5,10-methylene-tetrahydropteroylmono-, di-, and triglutamate substrates and metabolized (6R)-5,10-methylene-tetrahydro[3H]folate to the tetraglutamate derivative. Other folate derivatives were poor substrates or lacked activity. The specificity of the nucleotide site was wide. The magnesium salts of dATP, GTP, ITP, and UTP were effective alternate substrates for the reaction. The specificity of the glutamate binding site was very narrow. Of a wide variety of analogs tested, only L-homocysteate and 4-fluoroglutamate demonstrated affinity for the enzyme. Kinetic studies were consistent with an ordered Ter Ter mechanism with MgATP binding first to the enzyme, folate second, and glutamate last. The order of product dissociation from the enzyme was ADP, folate product, and Pi. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. The Michaelis constants for (6R)-5,10-methylene-tetrahydropteroyldiglutamate, the most effective folate substrate, MgATP, and L-glutamate were 2.3 microM, 5.6 mM, and 423 microM, respectively. Adenosine 5'-(3-thio)triphosphate and beta, gamma-methylene-ATP were inhibitors of the reaction and had higher affinities for the enzyme than ATP.

Adenosine Triphosphate↗
PubMed1983

Folylpoly-gamma-glutamate synthetases: properties and regulation.

The substrate specificities of folylpolyglutamate synthetases from Corynebacterium species, Lactobacillus casei, Streptococcus faecalis, and Chinese hamster ovary (CHO) cells are described. The purified Corynebacterium enzyme catalyzes the addition of glutamate moieties to reduced pteroylmonoglutamates, and to 5,10-methylenetetrahydropteroyldi- and triglutamates. The purified protein also possesses a separate dihydrofolate synthetase activity. Binding of pteroate substrates to dihydrofolate synthetase impairs folate binding to folylpolyglutamate synthetase. The in vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differs from source to source, appears to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources. Bacterial synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydropteroyl polyglutamates as their preferred, and sometimes only, polyglutamate substrates. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of the CHO cell synthetase. Products of one carbon metabolism do not affect the in vivo distribution of folylpolyglutamates in CHO cells. Although adenine changes the distribution of folylpolyglutamates in bacteria, this effect is mediated by changing the substrate availability for the synthetase reaction, and not by regulating the levels of the synthetase per se.

Animals↗
PubMed1983

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis.

Lactobacillus casei and Streptococcus faecalis accumulated labeled folic acid and metabolized this compound to poly-gamma-glutamates of chain lengths of up to 11 and 5, respectively. Octa- and nonaglutamates predominated in L. casei, and tetraglutamates predominated in S. faecalis. The most effective monoglutamate substrates for the L. casei and S. faecalis folylpoly-gamma-glutamate (folylpolyglutamate) synthetases were methylene- and formyltetrahydrofolate, respectively. Methylenetetrahydropteroylpoly-gamma-glutamates were the preferred poly-gamma-glutamate substrates for both enzymes and, in each case, the highest activity was observed with the diglutamate substrate. The final distribution of folylpolyglutamates in these bacteria appeared to reflect the ability of folates with various glutamate chain lengths to act as substrates for the bacterial folylpolyglutamate synthetases. The proportions of individual folylpolyglutamates were markedly affected by culturing the bacteria in medium containing adenine, whereas thymine was without effect. Adenine did not affect the level of folylpolyglutamate synthetase in either organism but caused a large increase in the proportion of intracellular folates containing one-carbon units at the oxidation level of formate, folates which are substrates for enzymes involved in purine biosynthesis. The folates with shorter glutamate chain lengths in bacteria cultured in the presence of adenine resulted from primary regulation of the de novo purine biosynthetic pathway, regulation which caused an accumulation of formyltetrahydropteroyl-poly-gamma-glutamates (folate derivatives that are ineffective substrates for folylpolyglutamate synthetases), and did not result from regulation of folylpolyglutamate synthetase per se.

Adenine↗
PubMed1982

Regulation of folylpoly-gamma-glutamate synthesis in mammalian cells. In vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Chinese hamster ovary cells.

Cultured Chinese hamster ovary cells accumulated labeled folate and 5-methyltetrahydrofolate and metabolized these compounds to polyglutamates of chain length up to 10, with hexa-, hepta-, and octaglutamates predominating. Intracellular vitamin was concentrated up to 50-fold, although the intracellular pteroylmonoglutamate concentration did not exceed that in the medium. Folylpolyglutamate levels in mutant cells (AUXB1), which are defective in the enzyme folylpolyglutamate synthetase, were less than 1.5% of wild type levels, while pteroylmonoglutamate levels were normal. Tetrahydropteroylpolyglutamates were the most effective polyglutamate substrates for the Chinese hamster ovary cell folylpolyglutamate synthetase. The final distribution of folylpolyglutamates in this cells appeared to be a reflection of the ability of different glutamate chain length folates to act as substrates for the synthetase. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing or lacking products of one carbon metabolism such as glycine, adenosine, or thymidine. However, these proportions were markedly influenced by the level of methionine in the culture medium. The possible significance of changes in the glutamate chain length of folates under different nutritional conditions in relationship to the regulation of one carbon metabolism is discussed.

Animals↗
PubMed1982

High performance liquid chromatography of folates: identification of poly-gamma-glutamate chain lengths of labeled and unlabeled folates.

A sensitive method is described for the identification of the polyglutamate chain lengths of labeled and unlabeled folates in biological extracts. Folates in bacterial or mammalian tissue extracts were quantitatively cleaved to rho-aminobenzoylpolyglutamates. The primary aromatic amines were converted to azo dyes of naphthylethylene diamine and were purified by chromatography on BioGel P2 polyacrylamide columns. The purified azo dyes were reductively reconverted to rho-aminobenzoylpolyglutamates, which were separated according to glutamate chain length by high performance liquid chromatography on a Partisil 10 SAX anionic exchanger. Unlabeled derivatives derived from tissue folates were detected and quantitated by their absorbance at 280 nm. Lactobacillus casei contained folates of glutamate chain length up to 11 with the octa- and nonaglutamates predominating, while tetraglutamates predominated in Streptococcus faecalis. Vitamin in rat liver was a mixture of pteroylmono- to heptaglutamate with the pentaglutamate predominating.

Animals↗
PubMed1982

Folate metabolism in the ethanol-fed rat.

The effect of long-term ethanol supplementation on folate metabolism was studied in McCollum rats receiving approximately 30% of their caloric intake as ethanol. Ethanol did not significantly affect hepatic or plasma total folate levels. The net hepatic uptake and metabolism of a labeled folate dose to polyglutamate forms were unimpaired by ethanol administration. In fact, the ethanol-fed animals metabolized hepatic pteroylmonoglutamates to polyglutamate forms, predominantly the pentaglutamate, at a slightly faster rate than the control animals, and the net hepatic uptake of the labeled dose was higher than in the controls. Tetrahydrofolate derivatives were the predominant one-carbon form of folate in the livers of ethanol-fed and control animals, although the proportion of folate in the 5-methyltetrahydrofolate form was slightly increased after ethanol supplementation. Although hepatic metabolism of folate appears to be unimpaired in the ethanol-fed rat, an increased rate of pteroylpolyglutamate synthesis, with a resulting increase in the tissue storage of folate, might be a contributing factor to the low serum folate levels reported in alcoholics.

Alcoholism↗
PubMed1982

Vitamin B-6 metabolism and turnover in the ethanol-fed rat.

The effect of long-term ethanol supplementation on vitamin B-6 metabolism and storage was studied in McCollum rats receiving approximately 30% of their caloric intake as ethanol. Hepatic pyridoxal 5'-phosphate levels were significantly depressed in the ethanol-fed animals. However, total hepatic vitamin B-6 levels were slightly higher than in control animals because of a marked increase in pyridoxamine 5'-phosphate levels. The metabolism of a labeled pyridoxine dose appeared unaffected by ethanol administration, with the final distribution of labeled metabolites, almost entirely pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate, mirroring that observed with unlabeled endogenous vitamin B-6. The daily turnover of total-body vitamin B-6 stores in ethanol-fed animals (2.7%) was lower than in control animals (3.8%). Whether this resulted from ethanol administration or a reduced vitamin B-6 intake was not ascertained. However, it was apparent that ethanol did not increase the turnover of total-body vitamin B-6 or decrease total-body stores of the vitamin, which were similar in both groups of animals on a body store per kilogram weight basis. Pyridoxic acid excretion reflected vitamin B-6 intake in both groups of animals and accounted for approximately 30% of the total excreted vitamin.

Alcoholism↗
PubMed1982

Folate derivatives in human cells: studies on normal and 5,10-methylenetetrahydrofolate reductase-deficient fibroblasts.

The distribution of folylpolyglutamates in normal and methylenetetrahydrofolate reductase-deficient human fibroblasts cultured in medium containing folic acid or 5-methyltetrahydrofolic acid has been determined. Human fibroblasts concentrated these folates to higher levels than in the medium, an effect that was more pronounced with methyltetrahydrofolate as the folate source. Over 95% of the intracellular vitamin derivatives were polyglutamates of chain length 2 to 10. The major derivatives were hexaglutamates in cells cultured with folic acid and heptaglutamates in cells cultured with methyltetrahydrofolic acid. No significant differences were detected in the polyglutamate distribution between normal and methylenetetrahydrofolate reductase-deficient fibroblast. Excess medium methionine reduced cell growth rates and intracellular vitamin levels and changed the predominant polyglutamate in cells cultured with methyltetrahydrofolate from hepta- to hexaglutamate. No significant differences were seen between the overall folate polyglutamate distributions of different one-carbon folate pools of normal fibroblasts, although slight changes in the proportions of individual polyglutamate forms were detected in the different pools.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗