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Methylenetetrahydrofolate dehydrogenase from the obligate anaerobic mesophile Clostridium formicoaceticum has been purified to homogeneity. It has a molecular weight of 60,000 ± 3,000 and consists of two subunits of equal molecular weight. The sedimentation constant s020, w is 3.8 S, the partial specific volume is 0.74 ml per g, and the Stokes radius is 33.2 A. The enzyme is specific for NAD, and with NADP there is no activity. NADP is only slightly inhibitory at concentrations above 10-2 m. The pure enzyme has an activity of 1,400 µmoles min-1 mg-1 at 37°, pH 7. It is active only with l, l-5,10-methylenetetrahydrofolate, and the d, l-isomer does not affect the reaction. The apparent Km for NAD is 7.9 x 10-4 m and for l, l-5,10-methylenetetrahydrofolate 6.6 x 10-5 m at 37° in 0.2 m potassium maleate, pH 7. The temperature optimum is about 45°. Below this temperature the enzyme exhibits a linear relationship in an Arrhenius plot, and the apparent activation energy is 8,500 cal mole-1.

Methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyltransferase were found to be localized in the soluble fraction of rat brain. They are clearly separated from serine hydroxymethyltransferase and the glycine cleavage complex which are localized in the mitochondria in this tissue. Hence, although the primary, if not the only, site of 5,10-methylenetetrahydrofolate formation in brain appears to be the mitochondrion, the utilization of this compound for 5-methyltetrahydrofolate synthesis and utilization of the latter compound for methylation of homocysteine occurs in the cytosol. Parallel experiments with rat liver confirmed that the reductase and homocysteine methyltransferase of this tissue are also localized in the soluble fraction, while the hydroxymethyltransferase is about evenly divided between mitochondria and cytosol. However, in liver (but not in brain) the reductase activity of the supernatant fraction is only partially expressed unless the fraction is dialyzed. We have found that this phenomenon, which initially suggested the occurrence of an endogenous inhibitor in liver extracts [Ordonez and Wurtman (1973) J. Neurochem., 21, 1447–1455] , is due to loss of the product (5, 10-methyl-enetetrahydrofolate) of the reductase assay via its oxidation by methylenetetrahydrofolate dehydrogenase, in combination with the NADP present in undialyzed extracts. All regions of the rat central nervous system tested contained methylenetetrahydrofolate reductase activity. Regional variations were observed however, with an almost threefold difference between the specific activities of the highest and lowest ranking regions Comparison of the rank order of 12 regions tested with respect to reductase specific activity (this study) and methyltetrahydrofolate-tryptamine N-methyltransferase specific activity [Mandell, Knapp, and Hsu (1974) Life Sci., 14, 1–17] shows a high positive correlation (r = 0.916) between these activities in the selected regions.

1. 1. The effects of dietary fructose, glucose, casein and fasting and oral folate upon the activity of four enzymes involved in folate metabolism [glutamate formimonotransferase (N-formimino-L-glutamate:tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5), serine hydroxymethyltransferase (L-serine: tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1), methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.5) and formyltetra-hydrofolate synthetase (formate;tetrahydrofolate ligase (ADP), EC 6.3.4.3)] were studied in the jejenum of rats. 2. 2. Individual sugars (fructose and glucose) have specific carbohydrate effects on glutamate formiminotransferase activity, with the activity being highest in the fructose-fed rats. With the three other enzymes, no differential effect was noted with the two sugars. The activities of three of the enzymes (glutamate formiminotransferase, serine hydroxymethyltransferase and methylenetetrahydrofolate dehydrogenese) were highest on the carbohydrate diets, less on the casein diet and least in the fasting state. In contrast, the activities of formyltetrahydrofolate synthetase were lowest in the carbohydrate-fed rats and significantly higher in the fasted and caseinfed rats. 3. 3. Oral folate (two different dosages) produced significant increases in the activities of three of the folate-metabolizing enzymes: glutamate formmiminotransferase, serine hydroxymethyltransferase and methylenetetrahydrofolate dehydrogenase. The activity of glutamate formiminotransferase increased with increasing amounts of oral folate, whereas the activities of the other two enzymes were the same on the two levels of oral folate. Oral folate had no significant effect upon the activity of formyltetrahydrofolate synthetase.

Glycollate metabolism in 5-day-old endosperm tissues of Ricinus communis L. was examined by feeding micromolar quantities of [2- 14 C]glycollate to tissue slices. It was found that glycollate carbon was rapidly incorporated into glyoxylate, glycine, serine, and carbon dioxide. Only small amounts of 14 C were incorporated into the sugars. Changes in the distribution of 14 C with time suggested that glyoxylate was a primary product and that glycine and serine were secondary products of glycollate metabolism. The results of feeding experiments are interpreted as indicating that a glycollate pathway leading to sugar biosynthesis is of minor importance compared to the rapid utilization of glycollate for the biosynthesis of glycine and serine. Enzymes necessary to catalyse the incorporation of glycollate into glycine and serine have been examined in castor-bean endosperm extracts. These included: glycollic acid oxidase, gloxylic acid reductase, glyoxylate transaminase, N 10 formyltetrahydrofolate synthetase, N 5 ,N 10 -methylenetetrahydrofolate dehydrogenase, and serine hydroxymethyltransferase.

The synthesis of several analogs of the title compound (Ia) which had (a) replacement of the 4-hydroxy group by thiol, (XXVIIIa), (b) replacement of the 6-methyl group by 6-phenyl (Ib), (c) replacement of the 2-amino group by 2-thiol (XXVI), or (d) replacement of both the 4-hydroxyl and 6-methyl groups by 4-thiol and 6-phenyl, respectively, have been described. The key intermediate was the appropriate 2,4,6-trisubstituted5-pyrimidyl-propionic acids which were synthesized by suitable transformation of the 5-pyrimidyl-propionitriles (VIII and XII). Replacement (a) or (b) gave an improvement in inhibition of 5, 10 - methylenetetrahydrofolate dehydrogenase, but the two changes in the same molecule (d) did not further increase inhibition. The 2-amino group of the title compound (Ia) can be replaced by 2-thiol without decreasing the inhibitory properties of Ia.

The N5,N10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activity of pea seedlings has been examined in cotyledon extracts during the first 7 days of germination. Specific enzyme activities increased over this period but were not appreciably changed in seeds which had imbibed chloramphenicol, cycloheximide and aminopterin solutions. The general properties of the enyzme from 18-day-old pea shoots were examined using preparations which ahd been fractionated with (NH4)2SO4 followed by chromatography on DEAE-cellulose. The enzyme was found to be relatively unstable after chromatography on DEAE-cellulose. Enzyme activity was maximal at pH 6·7 and had absolute requirements for formaldehyde and tetrahydrofolic acid. NADP was an effective hydrogen acceptor in the reaction although this coenzyme could be partially replaced by NAD. The enzyme was strongly inhibited by iodoacetate and p-chloromercuribenzoate and to a lesser extent by α,α′-dipyridyl and o-phenanthroline. The general properties of the plant enzyme are discussed in relation to those of the N5,N10-methylenetetrahydrofolate dehydrogenases of micro-organisms and animal tissues.

Methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) which is responsible for the enzymatic convertion of 5, 10-methylene-H4FA to 5, 10-methenyl-H4FA was found in various plant tissues. The enzyme was partially purified from pea seedlings and some of its properties were investigated. The enzyme was purified about 40-fold by fractionation with ammonium sulfate and gel-filtration. The optimum pH was 7.8. 5, 10-Methylene-H4FA and NADP were specifically required in the enzymatic reaction. Their Km values were 3.5×10-4M and 5.1×10-5M, respectively. Intracellular distribution of some folate-linked enzymes, i.e., methylenetetrahydrofolate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase, and phosphoribosylaminoimidazolecarboxamide formyltransferase, in pea seedlings were investigated. Major portions of these enzymes were located in the soluble fraction, while some activities of these enzymes were significantly detected in mitochondrial or microsomal fraction. A possible pathway for the synthesis and metabolism of folate coenzymes in plants is discussed.

5,10-Methylenetetrahydrofolate dehydrogenase was purified approximately 80-fold from lyophilized cells of Clostridium cylindrosporum. The purified enzyme is specific for TPN. 5,10-Methylenetetrahydropteroyltriglutamate and 5,10-methylenetetrahydropteroate, in addition to 5,10-methylenetetrahydrofolate, can serve as substrates. The Michaelis constants for TPN and 5,10-methylenetetrahydrofolate are 3.4 x 10-5 m and 2.6 x 10-5 m, respectively. Optimum enzyme activity is obtained at pH 6.8 to 8. The immediate product of the reaction is 5,10-methenyltetrahydrofolate, rather than 10-formyltetrahydrofolate. The equilibrium constant of the reaction is approximately 0.14. ATP at 2 mm inhibits the activity in crude extracts of Clostridium acidi-urici about 45%. The purified enzyme and the enzyme in crude extracts of C. cylindrosporum are less sensitive to inhibition by ATP.

In the different ways by which folic acid incorporates “one-carbon units” in nucleic acid synthesis, there are several enzyme-catalyzed steps at which various drugs are proposed to exert inhibition. In this article, dihydrofolate reductase, N 5 N 10 -methylenetetrahydrofolate dehydrogenase and tetrahydrofolate formylase have been investigated with regard to inhibition by phenobarbital, diphenylhydantoin, primidone, pyrimethamine and 2,4,7-triamino-6-phenyl-pteridine. Only the last two drugs mentioned, were found to inhibit dihydrofolate reductase by 50% at a concentration of about 4 · 10 −6 M . At no other instance could an inhibition be observed.

Two forms of methylenetetrahydrofolate dehydrogenase, separable by TEAE-cellulose chromatography, are present in wild type extracts of S.cerevisiae , one of which is absent in ad 3 mutant extracts.

Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP oxidoreductase EC 1.5.1.5) has been purified from Clostridium thermoaceticum, an obligate anaerobic thermophile. The enzyme has a molecular weight of 55,000 ± 5,000 and consists of 2 subunits of equal molecular weight. The sedimentation constant s20,w0 is 3.8 S, the partial specific volume 0.752 cc per g, and the Stokes radius 31.7 A. The enzyme appears homogeneous during Sephadex chromatography and ultracentrifugation, but during disc gel electrophoresis at pH 8.9 two bands of equal intensity are seen. Both bands are enzymatically active, and, when isolated individually, they again show the same two bands in the electrophoresis, indicating an equilibrium between the two forms. The enzyme has high thermal stability with a temperature optimum above 64° and exhibits a broken line in an Arrhenius graph. The apparent Km for NADP is 9 x 10-5 m and for methylenetetrahydrofolate 3.5 x 10-5 m. The enzyme is specific for NADP and NAD is a competitive inhibitor with a Ki of 2 x 10-4 m.

1. 1. The effects of oral sex hormones, dietary fructose and casein without and with actinomycin D or ethionine and fasting upon the activities of two folate-metabolizing enzymes (serine hydroxymethyltransferase ( L -Serine: tetrahydrofolate 5,10-hydroxymethyltransferase, E.C. 2.1.2.1) and methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP oxidoreductase, E.C. 1.5.1.5)) and two glycolytic enzymes (pyruvate kinase (ATP: pyruvate phosphotransferase, E.C. 2.7.1.40) and fructose diphosphate aldolase (fructose-1,6-diphosphate D -glyceraldehyde-3-phosphate lyase, E.C. 4.1.2.13)) were studied in the jejunum of female and male rats. 2. 2. Actinomycin, an inhibitor of DNA-dependent RNA synthesis, and ethionine, an inhibitor of protein synthesis, both effectively decreased the adaptive responses of jejunal glycolytic and folate-metabolizing enzymes to diet and oral sex hormones. These findings lend support to the hypothesis that stimulation of synthesis of certain RNA species is probably one of the primary mechanisms by which sex hormones (17β -estradiol and testosteron) and diet produce adaptive increases in the activities of certain jejunal glycolytic and folate-metabolizing enzymes.

In ad3 mutants, the specific activities for formyltetrahydrofolate synthetase and methenyltetrahydrofolate cyclohydrolase, represent ca. 10 % of wild type activity and the activity for methylenetetrahydrofolate dehydrogenase can represent 50 % of wild type activity under appropriate assay conditions.

A methotrexate-resistant subline of the Ehrlich ascites carcinoma contained approximately 14-fold more dihydrofolate reductase activity than did the drug-sensitive parent neoplasm. The activities of a number of tetrahydrofolate-dependent enzymes (i.e., of N 5 -formyltetrahydrofolate synthetase, N 5 , N 10 -methylenetetrahydrofolate dehydrogenase, N 5 -formyltetrahydrofolate isomerase, and serine hydroxymethylase) in cells of the resistant subline were similar to those of the sensitive neoplasm; thus, the increase in dihydrofolate reductase activity in the methotrexate-resistant cells was specific. Several properties of partially purified dihydrofolate reductase preparations from each cell line were similar, suggesting that no quantitative changes in the enzyme accompanied the transition to the resistant state. The elevated dihydrofolate reductase activity in the drug-resistant variant appeared to be sufficient to account for the refractoriness of this neoplasm, since considerable enzyme activity could be detected in these cells after large amounts of methotrexate. The problems of relating in vitro enzyme assays to events occurring in vivo are discussed.

1. 1. The effects of oral, intramuscular and conjugated sex hormones (progesterone, testosterone and estradiol-17β) upon the activities of four enzymes involved in folate metabolism, (glutamate formiminotransferase (N-formimino- L -glutamate:tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5), serine hydroxymethyltransferase ( L -serine : tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1), methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate : NADP oxidoreductase, EC 1.5.1.5) and formyltetrahydrofolate synthetase (formate : tetrahydrofolate ligase (ADP), EC 6.3.4.3)) were studied in the jejunum of normal and castrated male and female rats. 2. 2. These studies demonstrate that oral sex hormones (estradiol in females, testosterone in males) produce significant adaptive increases (P

The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A k was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 m m nicotinamide adenine dinucleotide phosphate (NADP + ) during purification; 0.01 m m EADP + with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at −20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP + -specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K m values for NADP + and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l , l -tetrahydrofolate) were calculated to be 0.021 and 0.026 m m , respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l -serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A k was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.