Polyglutamate Forms Research Articles

Effect of phenobarbitone on folic acid metabolism in the rat

Anticonvulsant drugs have well-known relationships with folic acid, often leading, in epileptic patients on long term treatment to signs of folic acid deficiency and to haematological complications. The findings of this work, carried out on rats, show strong interference of phenobarbitone with folic acid metabolism, particularly with pteroylpolyglutamate synthesis in liver. This is proved by lower polyglutamate content, lower 3H folate incorporation in polyglutamate forms and in protein-bound folates, and also by lower blood radioactivity, higher urinary excretion of 3H folate metabolites and by higher 3H folate concentration in kidneys.

Identification and cell level of folate derivatives from growing cultures of streptomycin-bleached Euglena gracilis

A general description of folate derivatives of growing cells of a streptomycin-bleached strain of Euglena gracilis Klebs strain Z Pringsheim has been obtained by combining data from differential microbiological assays on total or fractionated cell extracts with information from radiochromatographic analyses of folates from cells grown in [ 3H] folic acid or p-[ 14 C]- aminobenzoic acid. Lactobacillus casei assays, after treatment of the Euglena extracts with purified γ-glutamyl-carboxypeptidase, gave a value of about 60 μg/g dry wt. of folic acid. The distribution of the various forms of folates was 1% as formyl-monoglutamate, 8.6% as formyl di- and triglutamate, 25.9% as formyl-polyglutamate, 1.5% as methyl-monoglutamate, 10.4% as methyl di- and triglutamate and 52.4% as methyl-polyglutamate. No folates with an oxidized pteridine ring were detected. The preferred polyglutamate form was the hexaglutamate. Cell content, ratio between formyl and methyl derivatives, and percentage distribution in the mono-, oligo- and polyglutamate forms are compared with those of other organisms or tissues.

Methotrexate polyglutamates in cultured human cells.

Cultured human fibroblasts accumulated methotrexate polyglutamates to levels far in excess of the dihydrofolate reductase binding capacity. After four days in methotrexate-free medium the intracellular drug level dropped by 70% but nearly 80% of the remaining methotrexate was in the form of polyglutamates. Reduced folates prevented the accumulation of polyglutamates and the effects of methotrexate on deoxyuridine incorporation into DNA and cell growth if present along with methotrexate from the beginning of the incubation. However, the reduced folates were less effective if added to cells after a long exposure to methotrexate alone. Thymidine, glycine, and adenosine (GAT) prevent methotrexate toxicity only if maintained in the incubation medium. However, preincubation with methotrexate and GAT permits continued synthesis and accumulation of polyglutamates so that when the GAT and methotrexate were removed, toxicity from the retained methotrexate polyglutamates could be expressed. (2,4-diamino-5-(3'4'-dichlorophenyl)-6 methyl pyrimidine (DDMP), an antifol that does not form polyglutamate derivatives, inhibited deoxyuridine incorporation into DNA as long as the DDMP remained in the culture medium. Compared to what was seen with longer exposures to methotrexate, removal of DDMP resulted in a greater reversal of the inhibition of deoxyuridine incorporation.

Pharmacokinetics of erythrocyte methotrexate after high-dose methotrexate.

Erythrocyte methotrexate (MTX) concentrations were determined in 10 patients with metastatic osteogenic or soft tissue sarcoma after 52 cycles of high-dose methotrexate (HDMTX). In contrast to serum MTX, pharmacokinetics of erythrocyte MTX showed three distinct phases: A rapid decrease to a nadir 2-3 days after MTX was followed by a significant rise of erythrocyte MTX until days 10-14. Subsequently there was a third phase, with a definite decrease of erythrocyte MTX concentrations with half-lives of 30-40 days. Short-term repititions of HDMTX had considerable influence on the first two phases of the kinetics. Each curve surpassed that of the previous therapy, and erythrocyte MTX concentrations increased continuously to the values measured at the end of the HDMTX infusion. The following mechanisms of MTX enrichment in erythrocytes is discussed: In erythro- and normoblasts MTX is converted to polyglutamate forms, which are retained inside the cell and are probably the reason for the relatively high sensitivity to MTX. Upon resumption of erythropoiesis the release of freshly prepared erythrocytes containing MTX and predominantly MTX polyglutamates causes the renewed increase in blood MTX levels.

Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells.

To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines (MCF-7 and ZR-75-B) converted the drug to gamma-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 muM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH(2)-10-CH(3)-PteGlu(5) and 47 and 38% of the 4-NH(2)-10-CH(3)-PteGlu(4) remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 muM MTX and an additional 24 h in drug-free medium, [(3)H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [(3)H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 muM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 muM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates. These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

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 fibroblasts. 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 human fibroblasts, although slight changes in the proportions of individual polyglutamate forms were detected in the different pools.human cells fibroblasts folylpolyglutamates folate distribution methylenetetrahydrofolate reductase deficiency

The effect of derivatives of folic acid on the fluorodeoxyuridylate-thymidylate synthetase covalent complex in human colon xenografts

This study was designed to examine the endogenous concentrations of 5,10-methylenetetrahydrofolate ( CH 2 FH 4) in human colorectal adenocarcinoma xenografts, and to determine the ability of other folate derivatives to increase the formation of the ternary covalent complex between CH 2 FH 4, [6- 3 H]-5-fluorodeoxyuridylate (FdUMP) and thymidylate synthetase (TS, EC 2.1.1.45). Levels of CH 2 FH 4 were determined by measuring the release of [ 3H] 2O from [5- 3 H]-dUMP using TS from Lactobacillus casei. The reaction was linear from 1.9 × 10 −13 to 2.4 × 10 −11 mol of CH 2 FH 4 assayed. Concentrations of CH 2 FH 4 were low, ranging from 66 to 233 nM in cell water. Tetrahydrofolate ( FH 4) and dihydrofolate ( FH 2) increased complex formation, while 5-formyltetrahydrofolate (5- CHOFH 4) and 5-methyltetrahydrofolate (5- CH 3 FH 4) decreased the covalent binding of [6- 3 H]-FdUMP in vitro. Administration of FH 4 or FH 2 to tumor-bearing mice reduced subsequent formation of the covalent complex in vitro. Since 5- CH 3 FH 4 is a major derivative of folate in mammalian tissues, its effect on the covalent binding of [6- 3 H]-FdUMP was examined further; even in the presence of homocysteine and cyanocobalamin (B 12), the formation of the covalent complex was not increased. The fate of [5- 14 CH 3]- FH 4 was subsequently examined in vivo. In tumors at 1 hr after injection, 72% of the radiolabel remained as [5- 14 CH 3]- FH 4, while 17% had been converted to [ 14 C]-methionine or incorporated into protein. By contrast, however, the incorporation of radiolabel into the protein fraction of liver was almost 30-fold greater at this time. At 4 hr, radioactivity in tumors ( dpm/g) and in the fraction associated with [5- 14 CH 3]- FH 4 was decreased by over 60%, while metabolism was increased by only 13%. No polyglutamate forms of [5- 14 CH 3]- FH 4 were detected in tumors at 4 hr after treatment.

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.ethanol folate folylpoly-γ-glutamates

Accumulation of 5-methyltetrahydrofolate in cobalamin-deficient L1210 mouse leukemia cells.

Cobalamin-deficient L1210 mouse leukemia cells were propagated on [3H]folate under conditions designed to completely label the intracellular folate compounds. The latter, present almost entirely as polyglutamate forms, were converted to the corresponding monoglutamates by treatment with gamma-glutamylcarboxypeptidase; the monoglutamates were subsequently separated and identified by chromatography on Sephadex G-25. 5-Methyltetrahydrofolate accounted for approximately 70% of the total folate pool (17.8 nmol/10(9) cells), while the remainder was comprised largely of 5-formyltetrahydrofolate (13.0%), and tetrahydrofolate (9.5%). The identity of 5-methyltetrahydrofolate were further confirmed by chromatography on DEAE-cellulose and by its quantitative conversion to tetrahydrofolate after treatment with a highly purified preparation of methionine synthetase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13). In contrast, cobalamin-replete cells contained only 5.3% 5-methyltetrahydrofolate, along with 30% each of the other three reduced folate compounds. These results provide direct experimental proof of the "methyl trap" hypothesis, which predicted that 5-methyltetrahydrofolate should accumulate in cobalamin-deficient cells.

Folate polyglutamate and monoglutamate accumulation in normal and SV40-transformed human fibroblasts.

Folate polyglutamate and monoglutamate accumulation was measured in normal diploid and SV40-transformed human fibroblasts by Sephadex G-10 gel filtration chromatography. The cells were first depleted of folates and then provided with limiting amounts of [3H]-folic acid in order that the cells would accumulate only forms of folate necessary for proliferation. Both the normal and the transformed cells accumulated monoglutamate and polyglutamate forms, but by 72 hours of labeling the transformed cells contained 3-10 times more polyglutamate than the normal cells. The growth rates for the normal and transformed cells were similar at this limiting folic acid concentration. Thus, if folate polyglutamates are more important for the proliferation of SV40-transformed cells than the normal cells, then inhibition of polyglutamate formation may be an important potential target for chemotherapy.

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.

Tissue folylpolyglutamate chain-length characterization by electrophoresis as thymidylate synthetase-fluorodeoxyuridylate ternary complexes

Tissue folate polyglutamate chain lengths have been estimated by incorporation of the 5,10-methylenetetrahydrofolate polyglutamate form of the cofactor into ternary complexes with Lactobacillus casei thymidylate synthetase and [ 3H]fluorodeoxyuridylate. Complexes were separated electrophoretically on the basis of the charge associated with the free carboxyl function of each glutamate residue. The method is specific for methylenetetrahydrofolate and assures evaluation of chain length with minimal error introduced by pool equilibration or autooxidation. Introduction of formaldehyde into tissue extracts was used to convert tetrahydrofolate to the methylene derivative and show that the two pools have the same polyglutamate chain lengths. By this technique, mammalian liver tissue was shown to contain both pteroylpentaglutamate and pteroylhexaglutamate. Monkey, pig, and rabbit liver contain predominately hexaglutamate, mouse liver predominately pentaglutamate, and rat liver nearly equal proportions of each. Chicken liver contains mostly pentaglutamate and fish liver, hexaglutamate. The presence of penta-and hexaglutamates in L. casei and heptaglutamates in yeast confirms earlier observations by other methods and attests to the validity of this one.

Methotrexate polyglutamate synthesis by cultured human breast cancer cells.

We studied the conversion of methotrexate to poly-gamma-glutamyl derivatives by cultured human breast cancer cells. After incubation with 2 micro M [3',5',9-3H]methotrexate, MCF-7 cells were washed free of extracellular drug and were boiled to lyse cells and to release drug bound to dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The supernatant fraction was chromatographed on Sephadex G-15 to separate parent drug from polyglutamate forms. These cells rapidly and quantitatively converted methotrexate to polyglutamates, such that after 24 hr of incubation, 70 +/- (SEM) 3% of intracellular methotrexate existed as polyglutamates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase indicated that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. These studies demonstrate that methotrexate polyglutamates are readily formed in human tumor cells and bind to dihydrofolate reductase. Because these forms of the drug may be selectively retained within the cell, they may be important determinants of the duration of action and, ultimately, the cytotoxicity of methotrexate in human solid tumors.

Polyglutamate derivatives of folic acid coenzymes and methotrexate

Polyglutamate derivatives of the folate coenzymes are widely distributed in nature. Frequently, they are the predominant form of intracellular folate, and recent investigations have indicated that polyglutamates are the preferred substrates for most of the enzymes of folate metabolism. Dietary folates are mostly polyglutamate conjugates which must be broken down to the mono- or diglutamates before intestinal absorption can occur. Intracellularly, pteroyl monoglutamates are converted to the polyglutamate form, a process which appears to favor their cellular retention. Alterations in polyglutamate chain length may play an important role in the regulation of folate metabolism. Polyglutamate derivatives of the folate antagonist methotrexate have also been isolated. The synthesis and biological properties of these compounds along with their potential chemotherapeutic role are discussed.

87] Pteroylpolyglutamate synthase assay 1–5

This chapter discusses the basic concepts related to pteroylpolyglutamate synthase assay. Pteroylpolyglutamate synthases catalyze the formation of an amide bond between the γ-carboxyl group of a folate and the amino group of glutamic acid. The enzyme is determined by measuring the enzymic incorporation of radioactive glutamate into polyglutamate forms of folate on incubation with a folate substrate. Radioactive folates are separated from [ 14 C]glutamate by adsorption onto charcoal. After eluting the folates, the amount of radioactivity is determined. The assay described in the chapter was developed for use in the study of N. crassa mutants of me-6 and mac that appeared to be deficient in the ability to synthesize pteroylpolyglutamates. By the use of H 4 PteGlu 2 as well as H 4 PteGlu as the substrate, it was possible to identify the two components of the synthase of the wild-type organism. The assay also has been used in the laboratory in the study and partial purification of the enzyme of rat and calf liver. Identification of the products of synthase activity was not required and is not provided by this assay.

Folate Distribution in Cultured Human Cells

We have studied the distribution of folate coenzyme forms in cultured human fibroblasts from control lines and from lines derived from nine patients representing all of the published reports of 5,10-CH(2)-H(4)PteGlu reductase deficiency. Based on mobility on DEAE-Sephadex and differential microbiological assay the major folate fractions in extracts of human fibroblasts were 5-CH(3)-H(4)PteGlu, 10-CHO-H(4)PteGlu, and 5-CHO-H(4)PteGlu with smaller fractions, which included 5-CH(3)-H(2)PteGlu, 10-CHO-PteGlu, and H(4)PteGlu. Evidence that the 5-CHO-H(4)PteGlu may have been derived from 5,10-CH=H(4)PteGlu during extraction is presented. In most of the mutant fibroblasts the absolute concentration of 5-CH(3)-H(4)PteGlu was lower than in control cells but the proportion of intracellular folate which was 5-CH(3)-H(4)PteGlu was strikingly lower in mutant cells when determined by chromatography or differential microbiological assay. In both control and mutant cells most of the 5-CH(3)-H(4)-PteGlu was polyglutamate. The proportion of intracellular folate which was polyglutamate was similar in control and mutant cells. A direct relationship was observed between the proportion of cellular folate which was 5-CH(3)-H(4)PteGlu, and both the clinical severity of this disorder and the residual enzyme activity indicating that the distribution of different folates may be an important control of intracellular folate metabolism. These studies indicate that 5,10-CH(2)-H(4)PteGlu reductase is the only significant intracellular pathway for the generation of 5-CH(3)-H(4)PteGlu, that the activity of this enzyme regulates the level of this folate in control and mutant cells under conditions of culture used here, that the majority of intracellular folate is in the polyglutamate form, and that the relative distribution of folates may control folate metabolism by interaction in the various folate reactions.

Identification of folate binding proteins in rat liver.

Intraperitoneal injection of tritiated folic acid (PteGlu) into rats has revealed the presence of three separate protein fractions in the cytosol fraction of the liver and one in the mitochondria which bind folate derivatives. The proteins in the cytosol (cytosol I, II and III) have approximate molecular weights of 350,000, 150,000, and 25,000 and the protein in the mitochondria has an approximate molecular weight of 90,000 as estimated by gel filtration. The bound folate derivatives are primarily polyglutamate forms while cytosol II contains primarily bound 5-methyltetrahydrofolate polyglutamate derivatives. Little binding of radioactively labeled folic acid or 5-methyltetrahydrofolate to these fractions was observed when binding was carried out in vitro. Significant binding in vitro was observed, however, when a mixture of biosynthetically labeled natural folate derivatives was used. These proteins have not been purified, but cytosol III partially consists of the enzyme, tetrahydrofolate dehydrogenase (EC 1.5.1.3). Studies on the time course of folic acid incorporation into the liver showed that soon after injection nonmetabolized folic acid was bound to the plasma membrane fraction of the liver cell. It is suggested that at least one of the binding proteins in the cytosol may be involved in storage of the vitamin while the binding of nonmetabolized folic acid to the plasma membrane may reflect the existence of a carrier for folic acid transport into the cell.

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.

The availability of folates in man: effect of orange juice supplement on intestinal conjugase.

The mechanisms of the low availability of endogenous folates in orange juice and the inhibitory effects on the availability of pteroylheptaglutamate (PteGlu7) caused by orange juice supplements were investigated in man by the use of simulated orange juice (containing the principal components: citric acid, malic acid, etc.) with several different pHs. The endogenous folates in orange juice were identified as mainly 5-methyl reduced derivatives of pteroylpolyglutamates (40-45% pentaglutamate, 10-15% tetraglutamate, 30-40% monoglutamate, and the rest were tri-and diglutamates). The availability of PteGlu7 is decreased to 54% by supplementation with 600 g concentrated orange juice (pH 3.7) and to 39% and 66% by the supplement of 24 g and 12 g citric acid (pH 3.7), respectively. Neutralization of 12 g citric acid to pH 6.4 largely reverses the inhibitory effect. On the other hand, the availability of pteroylmonoglutamate (PteGlu) is not influenced by a supplement of orange juice or citric acid. The results suggest that the low availability of PteGlu7 is due to the inhibition of intestinal conjugase caused by the low pH. These observations suggest that pH of foodstuffs may be an important factor in the absorption of polyglutamate forms of folate.

DISTRIBUTION OF FOLIC ACID COENZYMES AND FOLATE DEPENDENT ENZYMES IN MOUSE BRAIN

—Folic acid coenzymes were found to be distributed equally between post-nuclear particulate and soluble fractions from whole Swiss mouse brain. Mitochondria isolated from the particulate fraction contained essentially only the N5-methyl derivative of folate, virtually all of which was in a polyglutamate form. Isolated synaptosomes contained significantly more folate than did mitochondria, with the greater proportion being non-N5-methyl derivatives. Osmotic lysis of synaptosomes released only a small portion of the folate; approximately 80 per cent remained with the particulate components of the synaptosome. The enzymes serine transhydroxymethylase and N5, N10-methylenetetrahydrofolate dehydrogenase were found in both the soluble and particulate fractions while formiminoglutamic acid:tetrahydrofolate formiminotransferase activity could not be detected. These findings may be of importance with respect to the synaptic functions of folate coenzymes, including the methylation of biogenic amines.