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1.
T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue.  相似文献   

2.
We developed a method for assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring their products of 5-methyltetrahydrofolate (5-CH(3)-H(4)folate) and tetrahydrofolate (H(4)folate) directly, using high-performance liquid chromatography with fluorescence detection. Folate derivatives and enzymes were stable in the assay process. No reagents in the assay mixture were found to disturb the separation and detection of both H(4)folate and 5-CH(3)-H(4)folate in our assay system. The detection limit of this method was less than 20 nM H(4)folate or 5-CH(3)-H(4)folate in the enzyme assay system. This analytical method, therefore, has a sensitivity high enough to obtain accurate parameters of Michaelis-Menten kinetics and for assays of crude extracts from various biological samples. In addition, the analytical procedure is very simple and economical; it may be a useful tool for studying methylenetetrahydrofolate reductase and methionine synthase activities.  相似文献   

3.
Effects of testosterone on the metabolism of folate coenzymes in the rat   总被引:1,自引:1,他引:0  
1. The effects of castration and testosterone treatment on enzymic activities involved in folate coenzyme metabolism in the liver and in accessory sex organs of male adult rats were studied. 2. In the liver of castrated rats the concentration of 10-formyltetrahydrofolate (10-HCO-H(4)folate) synthetase and tetrahydrofolate (H(4)folate) dehydrogenase were significantly decreased whereas that of 5,10-methylenetetrahydrofolate dehydrogenase increased; the treatment with five doses of testosterone caused a return to normal values of these activities. 3. In the prostate of castrated rats a pronounced decrease in H(4)folate dehydrogenase, serine hydroxymethyltransferase and 10-HCO-H(4)folate synthetase activities was observed. The administration of testosterone restored the enzymic activities to normal values. 4. In the seminal vesicles of castrated rats only 10-HCO-H(4)folate synthetase was markedly depressed; testosterone treatment not only restored activity to normal values but raised it to higher than normal values. The slight changes observed in other enzymic activities also returned to normal values with the hormone treatment. 5. These results are discussed in relation to a possible control mechanism of folate metabolism by testosterone.  相似文献   

4.
The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.  相似文献   

5.
To evaluate folate-dependent carbon incorporation into the purine ring, we measured (13)C-enrichment independently at C(2) and C(8) of urinary uric acid (the final catabolite of purines) in a healthy male after an independent oral dose of [6RS]-5-[(13)C]-formyltetrahydrofolate ([6RS]-5-H(13)CO-H(4)folate) or 10-H(13)CO-7,8-dihydrofolate (10-H(13)CO-H(2)folate). The C(2) position was (13)C-enriched more than C(8) after [6RS]-5-H(13)CO-H(4)folate, and C(2) was exclusively enriched after 10-H(13)CO-H(2)folate. The enrichment of C(2) was greater from [6RS]-5-H(13)CO-H(4)folate than 10-H(13)CO-H(2)folate using equimolar bioactive doses. Our data suggest that formyl C of [6RS]-10-H(13)CO-H(4)folate was not equally utilized by glycinamide ribotide transformylase (enriches C(8)) and aminoimidazolecarboxamide ribotide (AICAR) transformylase (enriches C(2)), and the formyl C of 10-H(13)CO-H(2)folate was exclusively used by AICAR transformylase. 10-HCO-H(2)folate may function in vivo as the predominant substrate for AICAR transformylase in humans.  相似文献   

6.
HPLC-EC has been used to measure the appearance of 5-CH3-H4 folic acid in human plasma following oral administration of folic acid. The process was found to be saturable in accordance with Michaelis-Menten kinetics. The apparent Km for this enzyme system indicates that low doses of oral folic acid are rapidly converted into 5-CH3-H4 folic acid, an observation consistent with the needs of intestinal absorption of essential trace nutrients. The appearance of L. casei active folate in plasma was not rate-limited and showed a biphasic relationship to dose. Preparative HPLC combined with L. casei bioassay demonstrated that most of the L. casei active folate appearing in plasma following a 20,000-micrograms dose of folic acid was due to the unmodified vitamin, only 5.6% being due to 5-CH3-H4 folic acid and with no detectable contribution from 5-CHO-H4 folic acid. The absorption characteristics of the system seem consistent between and within subject(s). No relationship could be demonstrated between predose levels of plasma 5-CH3-H4 folic acid and total folate in erythrocytes, which reflect the status of transport and storage forms of the vitamin, respectively.  相似文献   

7.
Production of folates by yeasts in Tanzanian fermented togwa   总被引:1,自引:0,他引:1  
We have investigated the impact of different yeasts and fermentation time on folate content and composition in a fermented maize-based porridge, called togwa, consumed in rural areas in Tanzania. The yeasts studied, originally isolated from indigenous togwa, belong to Issatchenkia orientalis, Pichia anomala, Saccharomyces cerevisiae, Klyveromyces marxianus and Candida glabrata. The main folate forms found, detected and quantified by HPLC during the fermentations were 5-methyl-tetrahydrofolate (5-CH(3)-H(4)folate) and tetrahydrofolate (H(4)folate). The content of H(4)folate, per unit togwa, remained fairly stable at a low level throughout the experiment for all strains, whereas the 5-CH(3)-H(4)folate concentration was highly dependent on yeast strain as well as on fermentation time. The highest folate concentration was found after 46 h of fermentation with C. glabrata (TY26) (6.91+/-0.14 microg 100 mL(-1)), corresponding to a 23-fold increase compared with unfermented togwa. The cell concentration per se could not predict the togwa folate level, as shown by the much higher specific folate content (g folate CFU(-1)) in the S. cerevisiae strain (TY08) compared with the other species tested. This study provides useful data when trying to maximize folate content in togwa as well as in other yeast-fermented products.  相似文献   

8.
Glycine N-methyltransferase (EC 2.1.1.20) was recently identified as a major folate binding protein of rat liver cytosol (Wagner, C., and Cook, R. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3631-3634). Activity of the enzyme is inhibited when the natural folate ligand, 5-methyltetrahydropteroylpentaglutamate (5-CH3-H4PteGlu5), is bound. It has been suggested that glycine N-methyltransferase plays a role in regulating the availability of methyl groups in the liver. Purified transferase was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. If 5-CH3-H4PteGlu5 was first bound to the transferase, phosphorylation was inhibited. Phosphorylation of glycine N-methyltransferase in vitro increased its activity approximately 2-fold. 5-CH3-H4PteGlu5 inhibited the activity of newly phosphorylated enzyme as well as native enzyme. Freshly isolated rat hepatocytes incorporated 32P-labeled inorganic phosphate into this folate binding protein. Chemical analysis of purified enzyme showed about 0.55 mol of phosphate present per mol of glycine N-methyltransferase subunit. These results indicate that phosphorylation of glycine N-methyltransferase may provide a mechanism for modulating the activity of this enzyme and support its role in regulating the availability of methyl groups.  相似文献   

9.
The composition of folate coenzymes in romaine lettuce was studied. Lettuce extract was purified on QAE-Sephadex A-25 and folate compounds were separated into a monoglutamate fraction and a polyglutamate fraction by chromatography on Sephadex G-15. Both the mono- and poly-glutamate fractions were resolved on DEAE-cellulose. Positive identification of DEAE peaks was made by further cochromatography with high specific activity radioactive marker folate compounds and with differential microbiological assay. The distribution of folate compounds in lettuce is as follows: 32% 5-CH3-H4PteGlu; 1% 5-CHO-H4PteGlu; 3% 5-CHO-H4PteGlu4; 9% 5-CH3-H4PteGlu4; 13% 5-CHO-H4PteGlu5; and 31% 5-CH3-H4PteGlu5.  相似文献   

10.
Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine. It is very abundant in liver cytosol and earlier studies have shown it to be present in high concentrations in the pancreas. We have previously reported that liver GNMT is allosterically inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-H4PteGlu5), and proposed that this represents a metabolic control mechanism which links the de novo synthesis of methyl groups to the methylating ability of the liver. We now report that pancreatic GNMT also contains bound folate in vivo. Purified pancreatic GNMT is inhibited by reduced folate polyglutamates in vitro. The KI for the synthetic (R,S)5-CH3-H4PteGlu5 is 2.4 x 10(-7) M. The natural (S) form of 5-CH3-H4PteGlu5 is tightly bound and has a Kd of 1.3 x 10(-7) M. One mole is bound per enzyme tetramer. These studies suggest that GNMT is important in the regulation of methyl group metabolism in the pancreas as well as in the liver.  相似文献   

11.
Folate metabolism in the rat was investigated using radiolabelled 5-methyltetrahydropteroylglutamate (5-CH3-H4PteGlu) and its oxidation products. 5-CH3-H4PteGlu is absorbed completely from the intestine, although in some preparations it is an equimolecular mixture of C-6 epimers, only one of which is naturally present in biological systems. The methyl group is incorporated into non-folate compounds, including methionine and creatine. No evidence was observed for the oxidation of the methyl group of 5-CH3-H4PteGlu to form other folate types. The tetrahydrofolate moiety of 5-CH3-H4PteGlu is metabolized in a similar manner to folic acid, forming formyl folates and tissue polyglutamates, and is catabolized by scission. The triazine oxidation product of 5-CH3-H4PteGlu is not metabolized by the rat or its gut microflora. 5-Methyl-5,6-dihydropteroylglutamate, however, is assimilated into the folate pool, but is substantially broken down by passage through the gut. The possible implication of this in scorbutic diets is discussed.  相似文献   

12.
R S Lipman  M S Jorns 《Biochemistry》1992,31(3):786-791
The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
A procedure involving solid-phase adsorption on bonded silica has been developed for trace enrichment and selective recovery of folate monoglutamates from liver tissue. A variety of reverse-phase (ethyl, octyl, octadecyl, phenyl) and anion-exchange (aminopropyl, quaternary amine, primary/secondary amine) cartridges were tested for their potential to adsorb and elute folate monoglutamates from standard solutions (50 nmol each of H4-pteroylglutamic acid (H4PteGlu), 5-CHO-H4PteGlu, 10-CHO-H4PteGlu, PteGlu, and 5-CH3-H4PteGlu). Quantitative recoveries were obtained from aminopropyl (-NH2) and all reverse-phase cartridges. For the analyses of rat liver folates, 20 ml of clear supernatant obtained from 5 g of tissue was treated with conjugase, which released folate monoglutamates from endogenous stores. Folate monoglutamates were then separated from nonfolate material by selective adsorption and recovery from -NH2 extraction cartridges. The procedure also provided a 10-fold concentrate, which allowed direct analysis by HPLC, using C-18 reverse-phase ion-pair columns coupled with uv detection (290 nm). Experiments with standard folates (n = 3) mixed with liver tissue and carried through the extraction, incubation, and trace-enrichment steps showed the following recoveries: 10-CHO-H4PteGlu, 55 +/- 5.0%; H4PteGlu, 80 +/- 5.0%; 5-CHO-H4PteGlu, 123 +/- 12.0%; and 5-CH3-H4PteGlu, 89 +/- 3.0%. Endogenous compositions of liver folates (n = 5) were as follows: 10-CHO-H4PteGlu, 1.03 +/- 0.3 nmol/g (6.7%); H4PteGlu, 5.70 +/- 1.0 (36.4%); 5-CHO-H4Pte Glu, 1.34 +/- 0.4 (8.7%); and 5-CH3-H4PteGlu, 7.34 +/- 1.2 (48.0%). Chromatographic peaks were identified by their retention times and by comparing their spectral profiles (obtained by a diode array detector) with respective pure folates. We found trace enrichment of biological folates on solid-phase extraction cartridges to be rapid and quantitative. The method allowed, for the first time, direct analysis of tissue folates by HPLC/uv methods.  相似文献   

14.
The dependence of the high-affinity transport systems for 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) and methotrexate on sodium ions and on pH was examined in freshly isolated rat hepatocytes. Previous studies indicated that transport of these folate derivatives was sodium-dependent. Experiments to determine the Km for sodium of 5-CH3-H4PteGlu transport showed no dependence on extracellular sodium. However, uptake was sodium-dependent when hepatocytes were preincubated for 30 min in sodium-free medium, a treatment which resulted in an increase in the transmembrane pH gradient (delta pH = pH out-pH in) and a decrease in the uptake of 5-CH3-H4PteGlu. Uptake of methotrexate displayed a linear dependence on extracellular sodium ions. Uptake of 5-CH3-H4PteGlu increased linearly as the transmembrane pH gradient decreased; i.e., as the medium became more acid with respect to the cytosol. Lineweaver-Burk and Scatchard plots of 5-CH3-H4PteGlu uptake indicated an apparent Km for H+ of about 24 nM, equivalent to a pH of 7.6. Hill-plots suggested a stoichiometry of 1:1 for the interaction of protons with the 5-CH3-H4PteGlu transport system. Both the Km and Vmax for 5-CH3-H4PteGlu transport were increased at pH 5.5 compared to pH 7.4, suggesting that extracellular protons increased the number of and/or the activity of the membrane carrier. In contrast, methotrexate transport was maximal at pH 7 where the transmembrane pH gradient was zero. These results suggest the possibility that 5-CH3-H4PteGlu may be cotransported along with H+ ions in hepatocytes, although they do not rule out a 'catalytic coupling' whereby protons interact with the carrier to stimulate substrate flux without concomitant H+ transport.  相似文献   

15.
16.
To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.  相似文献   

17.
T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.  相似文献   

18.
A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.  相似文献   

19.
The properties of folinate and 5-methyltetrahydrofolate (5-CH(3)-H(4)PteGlu) transport mechanism of Pediococcus cerevisiae were studied. The uptake was dependent on temperature, pH (optimum for both compounds at pH 6.0), and glucose. Iodoacetate, potassium fluoride, and sodium azide inhibited the uptake. 5-CH(3)-H(4)-PteGlu was apparently not metabolized but folinate was metabolized. Metabolism of folinate was reduced by preincubation of cells with fluorodeoxyuridine. The transport system for folinate and 5-CH(3)-H(4)PteGlu were specific for the l-isomers. Pteroylglutamate, aminopterin, and amethopterin did not interfere with the uptake. Tetrahydrofolate competed with the uptake of folinate. The transport of folinate and 5-CH(3)-H(4)PteGlu at 37 C conformed to Michaelis-Menten kinetics; apparent K(m) for both compounds was 4.0 x 10(-7)m, and the V(max) for folinate was 1.0 x 10(-10) moles per min per mg (dry weight) and for 5-CH(3)-H(4)PteGlu it was 1.6 x 10(-10) moles per min per mg (dry weight). Both compounds accumulated in the intracellular pool at a concentration about 80- to 140-fold higher than that in the external medium. Folinate inhibited competitively the uptake of 5-CH(3)-H(4)PteGlu with a K(i) of 0.4 x 10(-7)m. Unlike 5-CH(3)-H(4)PteGlu, which accumulated only at 37 C, folinate was also taken up at 0 C by a glucose- and temperature-independent process, which was not affected by the metabolic inhibitors mentioned above. Since at 0 C the intracellular concentration of folinate was also considerably higher than the external, binding of the substrate to some cellular component is assumed. The finding of an efficient transport system for l-5-CH(3)-H(4)PteGlu is of special interest, since this compound has no growth-promoting activity for P. cerevisiae.  相似文献   

20.
We have developed a novel HPLC-based fluorometric assay for serine hydroxymethyltransferase activity. In this assay, the 5,10-CH(2)-H(4)PteGlu formed by serine hydroxymethyltransferase activity is reduced to 5-CH(3)-H(4)PteGlu using NaBH(4). Then the fluorescent assay components are separated by reversed-phase chromatography under isocratic conditions and 5-CH(3)-H(4)PteGlu is quantified by comparison with standards. We show that this assay can be used to measure serine hydroxymethyltransferase activity at 10(-8) to 10(-3)M (6R,S)-H(4)PteGlu.  相似文献   

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