Folic acid compounds in romaine lettuce.

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.     

Characteristics of thymidylate synthase purified from a human colon adenocarcinoma

Thymidylate synthase has been purified greater than 4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH2-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH2-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu1-7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlu1 and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folypolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 microM) or CH2-H4PteGlu (4.3 microM) were detected when polyglutamates of [6R]CH2-H4PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGlu5. In addition, CH2-H4PteGlu4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH2-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH2-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.     

Trace enrichment of biological folates on solid-phase adsorption cartridges and analysis by high-pressure liquid chromatography

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.     

Mouse folylpoly-gamma-glutamate synthetase isoforms respond differently to feedback inhibition by folylpolyglutamate cofactors.

Folylpoly-gamma-glutamate synthetase (FPGS) is the enzyme responsible for metabolic trapping of reduced folate cofactors in cells for use in nucleotide and amino acid biosynthesis. There are two isoforms of FPGS expressed in mouse tissues, one is expressed in differentiated tissue, principally liver and kidney, and the other in all rapidly proliferating cell types. The present study sought the functional difference that would explain the evolution of two mouse FPGS species. Recombinant cytosolic mouse isozymes were compared with respect to steady state kinetics, chain length of polyglutamate derivatives formed, and end-product inhibition by the major reduced folylpentaglutamate cofactors. Both isoforms were equally effective in catalyzing the addition of a mole of glutamic acid to reduced folate monoglutamate substrates. Each isoform was also capable of forming long chain polyglutamate derivatives of the model folate, 5,10-dideazatetrahydrofolate. In contrast, the FPGS isoform derived from rapidly proliferating tissue was much more sensitive to inhibition by (6R)-5,10-CH(2)-H(4)PteGlu(5) and (6S)-H(4)PteGlu(5) than the isoform expressed in differentiated tissues, as demonstrated by 13- and 6-fold lower inhibition constants (K(i)), respectively. Interestingly, each isozyme was equally sensitive to inhibition by (6R)-10-CHO-H(4)PteGlu(5). We drew the conclusion that the decreased sensitivity of the FPGS expressed in mouse liver and kidney to feedback inhibition by 5,10-CH(2)-H(4)PteGlu(5-6) and H(4)PteGlu(5-6) may have evolved to permit accumulation of a larger folate cofactor pool than that found within rapidly proliferating tissue.     

Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer

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.     

Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates

We have studied the effects of methotrexate (MTX-Glu1) and the polyglutamate derivatives of methotrexate (MTXPGs) with 2, 3, 4, and 5 glutamyl residues on the catalytic activity of thymidylate synthase purified from MCF-7 human breast cancer cells and on the kinetics of the ternary complex formation by 5-fluoro-2'-deoxyuridine 5'-monophosphate, folate cofactor, and thymidylate synthase. MTX-Glu1 exhibited uncompetitive inhibition of thymidylate synthase when reaction kinetics were analyzed by either double reciprocal plots or a computerized mathematical model based on nonlinear least-squares curve fitting. The Ki for MTX-Glu1 inhibition was 13 microM and the I50 was 22 microM, irrespective of the degree of polyglutamation of the folate. In contrast, the polyglutamated derivatives of MTX all acted as noncompetitive inhibitors. The MTXPGs had 75-300-fold greater potency than MTX-Glu1 as inhibitors of thymidylate synthase catalytic activity, with Ki values from 0.17 to 0.047 microM for MTX-Glu2 to MTX-Glu5, respectively. Neither MTX-Glu1 nor MTXPGs promoted the formation of a charcoal-stable ternary complex with thymidylate synthase and 5-fluoro-2'-deoxyuridine 5'-monophosphate. CH2-H4PteGlu5 (where PteGlu represents pteroylglutamic acid) was found to be 40-fold more potent than CH2-H4PteGlu1 in participating in the formation of a ternary complex, and 10 microM MTX-Glu5 significantly inhibited the formation of a ternary complex containing this folate as cofactor. The inhibition was determined to be due to a reduction in the kon. The potency of this inhibition was markedly greater in the presence of CH2-H4PteGlu1 as compared to CH2-H4PteGlu5. This finding suggests that the degree of interference with complex formation in intact cells would depend on the state of polyglutamation of available folate cofactor. Ternary complex formation with H2PteGlu5 as the folate cofactor was also investigated, and a 50% reduction in complex formation was found in the presence of a 2 microM concentration of MTX-Glu5. These findings have significant implications regarding the mechanism of action of MTX-Glu1 and contribute to an understanding of the complex interactions of MTX-Glu1 and 5-fluorouracil.     

The metabolism of 5-methyltetrahydropteroyl-L-glutamic acid and its oxidation products in the rat.

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.     

A sensitive radioenzymatic assay for (S)-5-formyltetrahydrofolate.

A highly sensitive, radioenzymatic method has been developed for the specific and quantitative estimation of (S)-5-formyltetrahydrofolate. This method is based on enzymatic cycling of the 5-formyl derivative to methylenetetrahydrofolate followed by entrapment into a stable ternary complex with thymidylate synthase and tritiated fluorodeoxyuridylate. Determination of bound radiolabeled ligand permits estimation of the original folate. The initial cycling step is catalyzed by the enzyme, methenyltetrahydrofolate synthetase, which is specific for the (S)-diastereomer of 5-formyltetrahydrofolate and generates a product which can be further cycled to tetrahydrofolate using either 10-formyltetrahydrofolate deacylase or glycinamide ribonucleotide transformylase. Tetrahydrofolate is ultimately converted to the entrapable methylene derivative in the presence of excess formaldehyde. Using this assay recovery of reference (S)-5-formyltetrahydrofolate was linear over the range 0.03-1.9 pmol with an average recovery of 83 +/- 2%. The method has been applied to estimation of plasma (S)-5-formyltetrahydrofolate from a volunteer who had been administered (R,S)-5-formyltetrahydrofolate. Where comparison was possible, estimation of plasma (S)-5-formyltetrahydrofolate by this one step ternary complex-based method yielded results that were very similar to those observed by Straw et al. (Cancer Res., 44, 3114, 1984) who used an HPLC-based method for separation of diastereomeric mixtures of reduced folates and microbiological growth dependence to determine (S)-5-formyltetrahydrofolate.     

Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver

Thymidylate synthase has been purified 1700-fold from fetal pig livers by using chromatography on Affigel-Blue, DEAE-52, and hydroxylapatite. Steady-state kinetic measurements indicate that catalysis proceeds via an ordered sequential mechanism. When 5,10-methylenetetrahydro-pteroylmonoglutamate (CH2-H4PteGlu1) is used as the substrate, dUMP is bound prior to CH2-H4PTeGlu1, and 7,8-dihydropteroylmonoglutamate (H2PteGlu1) is released prior to dTMP. Pteroylpolyglutamates (PteGlun) are inhibitors of thymidylate synthase activity and are competitive with respect to CH2-H4PteGlu1 and uncompetitive with respect to dUMP. Inhibition constants (Ki values), which correspond to dissociation constants for the dissociation of PteGlun from the enzyme-dUMP-PteGlun ternary complex, have been determined for PteGlun derivatives with one to seven glutamyl residues: PteGlu1, 10 microM; PteGlu2, 0.3 microM; PteGlu3, 0.2 microM; PteGlu4, 0.06 microM; PteGlu5, 0.10 microM; PteGlu6, 0.12 microM; PteGlu7, 0.15 microM. Thus, thymidylate synthase from fetal pig liver preferentially binds pteroylpolyglutamates with four glutamyl residues, but derivatives with two to seven glutamyl residues all bind at least 30-fold more tightly than the monoglutamate. When CH2-H4PteGlu4 is used as the one carbon donor for thymidylate biosynthesis, the order of substrate binding and product release is reversed, with binding of CH2-H4PteGlu4 preceding that of dUMP and release of dTMP preceding release of H2PteGlu4. Vmax and Km values for dUMP and CH2-H4PteGlun show relatively little change as the polyglutamate chain length of the substrate is varied.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo characterization of the absorption and biotransformation of pteroylmonoglutamic acid in man: a model for future studies

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.     

Carrier-Mediated Transport of Folate in a Mutant of Pediococcus cerevisiae

A mutant strain of Pediococcus cerevisiae (P. cerevisiae/PteGlu) was isolated which grows on low-folate (PteGlu) concentrations (200 pg/ml). The growth response of the parent and mutant strains to folinate (5-CHO-H(4)PteGlu) was the same. The transport of (14)C-PteGlu by P. cerevisiae/PteGlu was temperature-dependent (Q(10) between 27 C and 37 C was about 2), energy-dependent, and pH-dependent and was inhibited by iodoacetate, 2,4-dinitrophenol, potassium fluoride, and sodium azide. The uptake obeyed saturation kinetics with an apparent K(m) of 6.6 x 10(-6) M and V(max) of 4.0 x 10(-10) mol per min per mg (dry weight). At the steady state the intracellular concentration of PteGlu was 120-fold higher from that of the medium. Reduced folates like 5-CHO-H(4)PteGlu and methyl-tetrahydrofolate (5-CH(3)-H(4)PteGlu) as well as 2,4-diaminoanalogues (amethopterin and aminopterin) were shown to compete for the PteGlue-carrier.     

Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism

Most mammalian cells receive exogenous folate from the bloodstream in the form of 5-methyltetrahydropteroylmonoglutamate (CH3-H4PteGlu1). Because this folate derivative is a very poor substrate for folylpolyglutamate synthetase, the enzyme that adds glutamyl residues to intracellular folates, CH3-H4PteGlu1 must first be converted to tetrahydropteroylmonoglutamate (H4PteGlu1), 10-formyltetrahydropteroylmonoglutamate (CHO-H4PteGlu1), or dihydrofolate (H2folate), which are excellent substrates for folylpolyglutamate synthetase. Polyglutamylation is required both for retention of intracellular folates and for efficacy of folates as substrates for most folate-dependent enzymes. Two enzymes are known that will react with CH3-H4PteGlu1 in vitro, methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyltransferase (cobalamin-dependent methionine synthase). These studies were performed to assess the possibility that methylenetetrahydrofolate reductase might catalyze the conversion of CH3-H4PteGlu1 to CH2-H4PteGlu1. CH2-H4PteGlu1 is readily converted to CHO-H4PteGlu1 by the action of methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, and these enzyme activities show very little preference for folypolyglutamate substrates as compared with folylmonoglutamates. We conclude from in vitro studies of the enzyme that methylenetetrahydrofolate reductase cannot convert CH3-H4PteGlu1 to CH2-H4PteGlu1 under physiological conditions and that uptake and retention of folate will be dependent on methionine synthase activity.     

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.     

Purification and properties of pancreatic glycine N-methyltransferase.

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.     

Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.     

Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis

Site-directed mutagenesis was used to characterize the functional role of lysine-411, a conserved amino acid located in putative transmembrane domain (TMD) 11 of the human reduced folate carrier (hRFC). Lysine-411 was mutagenized to arginine, glutamate, and leucine, and the mutant constructs (K411R-, K411E-, and K411L-hRFC, respectively) were transfected into hRFC-deficient K562 cells. The mutant hRFC constructs were all expressed at high levels and restored 22-36% of the methotrexate (MTX) transport level in wild-type (K43-6) hRFC transfectants. Although 5-formyl tetrahydrofolate (5-CHO-H(4)PteGlu) uptake levels for both the K411E- and K411L-hRFCs were also impaired (approximately 33% and 28%, respectively), a complete restoration of the wild-type level was observed for K411R-hRFC. While loss of MTX transport activity for the K411R-hRFC transfectant was associated with an incomplete restoration of MTX sensitivity compared to K43-6 cells, these cells were similarly sensitive to Tomudex. The K411R-hRFC transfectants showed an approximately threefold decreased growth requirement for 5-CHO-H(4)PteGlu compared to K43-6 cells. The 5-CHO-H(4)PteGlu transport stimulation observed for the wild-type carrier in chloride-free buffer was also observed for K411R-hRFC, however, this response was decreased for the K411E- and K411L-hRFCs. The preservation of low levels of transport for the K411E- and K411L-hRFCs suggest that the amino acid at position 411 does not directly participate in the binding of anionic hRFC substrates. However, a functionally important role for a basic amino acid at position 411 was, nonetheless, implied by the increased MTX transport for wild-type hRFC over the K411 mutant hRFCs, and the highly selective uptake of 5-CHO-H(4)PteGlu over MTX for K411R-hRFC.     

Phosphorylation modulates the activity of glycine N-methyltransferase, a folate binding protein. In vitro phosphorylation is inhibited by the natural folate ligand

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.     

An HPLC-based fluorometric assay for serine hydroxymethyltransferase

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.     

Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae

In this study, we investigated methionine synthase from Candida albicans (CaMET 6p) and Saccharomyces cerevisiae (ScMET 6p). We describe the cloning of CaMet 6 and ScMet 6, and the expression of both the enzymes in S. cerevisiae. CaMET 6p is able to complement the disruption of met 6 in S. cerevisiae. Following the purification of ScMET 6p and CaMET 6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 108, 84, 95, and 13 microM, respectively. The Michaelis constants for CaMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 113, 129, 120, and 14 microM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) as a substrate. We conclude that ScMET 6p and CaMET 6p require a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to the bacterial metE homologs. The cloning, purification, and characterization of these enzymes lay the groundwork for inhibitor-design studies on the cobalamin-independent fungal methionine synthases.     

Na+ and pH dependence of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes

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.