Tetrahydrofolate Dehydrogenase Research Articles

The effects of folic acid supplementation during pregnancy in the rat.

1. Twenty-four Sprague-Dawley female rats were randomly assigned to three groups (groups A, B, C). Group A was given a folic acid-free diet and groups B and C received 0.0018 g folic acid/kg diet. Rats in group C were also given a supplement of 1 mg folic acid/d by intraperitoneal injection. 2. After 14 d of feeding the rats were mated. The diets were continued throughout gestation. On day 21, of gestation the dams were killed and their livers and products of conception assayed for RNA, DNA, protein and tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate dehydrogenase; EC 1.5.1.3) activity. 3. The foetuses, placentas and livers from supplemented rats (group C) were significantly larger than those from groups A and B and had a higher content of RNA, DNA and protein. Those tissues from group A dams were smaller than those from the other groups and had a correspondingly reduced nucleic acid and protein content. 4. The activity of tetrahydrofolate dehydrogenase, the first and rate-limiting enzyme in the metabolism of folate, was increased in the folate supplemented rats (group C) and reduced in the rats given a folic acid-free diet (group A). These changes in enzyme activity could explain the differences in nucleic acid biosynthesis and growth shown by the different groups.

Tetrahydrofolate dehydrogenase in blood cells and in hematopoietic organs of different vertebrate species

Some general aspects in cytochemical demonstration of the tetrahydrofolate dehydrogenase, concerning the final reaction product, are studied. Steady and variable factors are detected in a comparative study of Vertebrate hemopoiesis: the enzyme exhibits peculiar features in different cell types. The reactivity progressively decrease in the erythropoietic series, in concomitance with hemoglobin synthesis. Conversely an increase in the intensity of reaction is found in the granulocytopoietic series; the correspondence of positive material with the specific (eosinophil and heterophil) granulations can be discussed. The thrombocytopoietic series is also labelled by this reaction.

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.

The role of tetrahydrofolate dehydrogenase in the hepatic supply of tetrahydrobiopterin in rats.

The reduction of 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin by rat liver tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is competitively inhibited by trimethoprim lactate (apparent Ki 0.285 muM). An apparent Michaelis constant of 43 muM for dihydrobiopterin was obtained, which is 430 times higher than the reported Km for dihydrofolate with this enzyme. The reduction of dihydrobiopterin is thus more susceptible to inhibition by trimethoprim lactate than is the reduction of dihydrofolate. However, intraperitoneal administration of trimethoprim had no significant effect on the hepatic supply of tetrahydrobiopterin in rats.

The regulation of the degradation of methionine and of the one-carbon units derived from histidine, serine and glycine

The isolated perfused rat liver and suspensions of isolated hepatocytes readily metabolize histidine but contrary to the liver in vivo do not form glucose. Formiminoglutamate was identified as a major product of histidine degradation in the isolated tissue preparations. The accumulation of formiminoglutamate was much diminished by the addition of physiological concentrations (0.1 m m) of l-methionine. No other protein amino acid had the same effect but l-ethionine was equally effective. Methionine, like other amino acids and low molecular cell constituents, is washed out of the liver cells on perfusion and on suspension in semi-synthetic media containing no amino acids. Addition of methionine restores the physiological tissue concentration. An accumulation of formiminoglutamate is already known to occur on histidine loading in vitamin B 12 deficiency. Thus the isolated liver preparations behave in this respect like the B 12-deficient liver in vivo. In vitamin B 12 deficiency the synthesis of methionine from homocysteine is impaired because of the cofactor function of vitamin B 12 in the methylation of homocysteine. The common feature of B 12-deficient liver and the liver preparations used is thus a deficiency in methionine. The degradation of formiminoglutamate involves the transfer of the formimino group to tetrahydrofolate and the conversion of the one-carbon unit to CO 2. An intermediate in this reaction is formyl tetrahydrofolate. The analysis of the methionine effect revealed that the release of CO 2 from formate by hepatocytes (which involves as an intermediary step the synthesis of formyl tetrahydrofolate) is also promoted by methionine or ethionine. The data are in accordance with the conclusion that the point of action of methionine is formyl tetrahydrofolate dehydrogenase which is activated by this amino acid. This activation may be looked upon as one of the key factors (there are others to which reference is made) in the regulation of folate metabolism and of methionine metabolism. Two pathways are open to the one-carbon units attached to tetrahydrofolate derived mainly from histidine, serine and glycine: either disposal by dehydrogenation to CO 2 or preservation for the supply of methyl groups for the regeneration of methionine from homocysteine, formyl groups in purine synthesis and of the one-carbon unit needed for the methylation of deoxyuridine monophosphate. Thus regulation of degradation versus preservation is necessary. This implies that there must be a flexible outlet disposing of surplus one-carbon units. Otherwise free tetrahydrofolate would disappear by complete conversion into its one-carbon derivatives. This outlet is the formyl tetrahydrofolate dehydrogenase reaction. The outlet must be open when there is excess one-carbon units over demand and blocked when the supply is limiting. The indicator of excess of supply is the concentration of methionine; the activation by methionine of formyl tetrahydrofolate dehydrogenase opens up the pathway by which the excess is degraded. In the case of methionine also regulation of degradation versus preservation is necessary. As methionine is an essential amino acid adequate amounts for protein synthesis and the methyl carrier function must be preserved while excess is removed to give energy and/or glucose. The branching point at which the fate of the carbon skeleton of methionine is decided is the stage of homocysteine. If this is not methylated it reacts with serine to enter the cystathionine pathway. The signal deciding the fate of homocysteine in favor of degradation is a decreased supply of methyl tetrahydrofolate. When the cell is saturated with methionine formyl tetrahydrofolate dehydrogenase becomes activated and this decreases the generation of methyl tetrahydrofolate so that homocysteine remains unmethylated and forms cystathionine. Thus the activation of formyl tetrahydrofolate dehydrogenase by methionine at the same time regulates the metabolism of one-carbon units and of methionine, by facilitating the irreversible disposal of one-carbon units and of the carbon skeleton of methionine. Whether activation of formyl tetrahydrofolate dehydrogenase by methionine is a direct effect of methionine on the enzyme or is brought about indirectly is an open question. This work started with an analysis of the observation that isolated liver preparations, contrary to the liver in vivo, form no glucose from histidine. It led to one major new observation, namely that the fate of the one-carbon units attached to tetrahydrofolate depends on the concentration of methionine. This finding proved relevant to the understanding of regulatory mechanisms, and so the work ended by arriving at new concepts about the regulation of the metabolism of methionine and of the one-carbon units handled by tetrahydrofolate. The results bear on several observations and concepts recorded in the literature, such as the methyl folate trap hypothesis.

Competitive protein binding assay for methotrexate.

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran--albumin coated charcoal. Scatchard plot analysis of the enzyme--drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant Ka of 2.1 X 10(8) M-1. This high affinity binding permits detection of methotrexate in the range of 0.3--30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5-methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5-formyl tetrahydropteroyl-glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4-amino-4-deoxy-10-methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.

Gene expression of foreign metaphase chromosomes introduced into cultured mammalian cells?

Transfer of genetic information from isolated hamster chromosomes to mouse cells is described. Metaphase chromosomes isolated from Chinese hamster diploid cells were incubated with mouse Cl. 1-d cells deficient in thymidine kinase activity. Two viable colonies appeared from the treated mouse cells after HAT selection with a frequency of about 10 −8. The first colony isolated (Cl. 1) failed to grow, however. The second colony isolated (Cl. 2) grew well in HAT medium and was subcultured for more than 70 generations. Cl. 2 cells possessed an elevated tetrahydrofolate dehydrogenase activity of molecular species resembling that of Chinese hamster cells, as shown by disc electrophoresis. The cell line also expressed surface antigen(s) specific to hamster species, as shown by mixed hemadsorption test and immune cell electrophoresis. This latter phenotype disappeared after prolonged cultivation (59 generations) of the cells in non-selective medium. The karyotype of Cl. 2 cells corresponded to that of the mouse species and was quite different from that of hamster cells. Hamster chromosomes could not be identified in any of the cell clones by detailed analysis by the banding method (Q- and C-band). Not one revertant cell was obtained among 4.2×10 8 Cl. 1-d cells in the control.

Regulation of one-carbon biosynthesis and utilization in Escherichia coli.

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10-methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.

Diurnal variations of liver folate metabolism in rats maintained under controlled feeding schedules.

SummaryRats maintained under controlled feeding schedules exhibit daily rhythms of the enzyme activities involved in folate metabolism. Tetrahydrofolate dehydrogenase and serine hydroxymethyl transferase show only small variations in their activity in the range of the 24-hr cycle. The 5,10-CH2-H4folate dehydrogenase shows a peak of activity at 6 PM (18:00), while the 5,10-CH2-H4folate synthetase activity reaches its lowest level between 3:00 PM (15:00) and 3:00 AM (3:00). The liver content of folate coenzymes does not change significantly during the whole day.

Effects of steroids, folate deprivation and protein deprivation upon tetrahydrofolate dehydrogenase levels in mammalian tissues

The effects of sex hormones, glucocorticoids, folate and protein deprivation upon tetrahydrofolate dehydrogenase activity in mammalian tissues were studied. In male rats estradiol induced significant increase in the activity of jejunal tetrahydrofolate dehydrogenase but only a slight increase occurred in the jejenum of hormone-treated female counterparts. In both sexes enzyme activity in liver and spleen was not altered by estradiol administration. Under the influence of testosterone, activity of hepatic tetrahydrofolate dehydrogenase fell to 50% of the normal level in male animals while a significant rise in the enzyme activity occurred in the jejunum. In female rats androgen administration had no significant effect upon the enzyme activity in liver, spleen or jejunum. Cortisone or prednisolone did not induce any changes in the activity of tetrahydrofolate dehydrogenase in liver or in spleen, but these steroids induced significant increases in the activity of the enzyme in rat but not in mouse jejunum. Under conditions of folate and/or protein deprivation or fasting, no significant changes were observed in the activity of tetrahydrofolate dehydrogenase in all tissues studied.

Improved purification of tetrahydrofolate dehydrogenase from L1210 leukemia by affinity chromatography

Tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.3; formerly known as dihydrofolate reductase) from a high enzyme mutant of L1210 mouse leukemia was purified to homogeneity by a simple two step procedure involving pH 5.1 precipitation of inert protein, and affinity chromatography employing a methotrexate-agarose column. By raising the pH and ionic strength of the eluting buffer from 0.05 M citrate (pH 6.0) to 0.05 M Tris-HCl (pH 8.5) containing 0.1 M KCl, a peak was eluted containing pure enzyme (spec. act. 2800 μmoles/h per mg). Purity was confirmed by methotrexate titration and polyacrylamide disc electrophoresis. A second small peak containing tetrahydrofolate dehydrogenase activity was eluted by further increasing the pH of the 0.05 M Tris-HCl buffer to 9.0, and increasing the KCl concentration to 0.4 M. The material in this peak showed an absorbance at 258 nm, suggesting that a nucleotide was bound to the enzyme. The identify of this material has yet not been established.

Purification of tetrahydrofolate dehydrogenase by affinity chromatography

Tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.3; formerly known as dihydrofolate reductase) from a subline of the L1210 lymphoma has been purified to apparent homogeneity with high recovery by simple steps using agarose to which the enzyme inhibitor amethopterin was coupled. A crude (NH 4) 2SO 4 precipitate was applied to a column of methotrexate-agarose which was washed with buffer containing NADPH to increase the binding of the enzyme to the column, and to elute non-enzyme protein. Buffer at high pH, containing dihydrofolate and high concentration of salt then eluted the enzyme together with some nucleic acid contaminants which were removed on a column of Sephadex G-75.

Effects of testosterone on the metabolism of folate coenzymes in the rat.

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.

Studies on the Regulation of One-Carbon Metabolism: THE EFFECTS OF FOLATE CONCENTRATION IN THE GROWTH MEDIUM ON THE ACTIVITY OF THREE FOLATE-DEPENDENT ENZYMES IN LACTOBACILLUS CASEI

Abstract The specific activities of 10-formyl tetrahydrofolate synthetase, dihydrofolate reductase, and 5,10-methylene tetrahydrofolate dehydrogenase in extracts of Lactobacillus casei increase during the period of logarithmic growth of the culture and decrease during the stationary phase. The rise in specific activities may indicate a rapid rate of synthesis since it is prevented by actinomycin D and chloramphenicol. The decrease in activity in the extracts of cells harvested during the stationary phase does not appear to result from the occurrence of proteolytic activity or from incomplete liberation. Variations in the concentration of folate in the growth medium have two effects on the specific activities of the enzymes. Extracts of organisms grown in the presence of 1.4 x 10-9 m folate have higher specific activities of 10-formyl tetrahydrofolate synthetase, dihydrofolate reductase, and 5,10-methylene tetrahydrofolate dehydrogenase than extracts from organisms grown in media containing lower levels of folate. This difference may be related to alterations in the growth rate of the bacteria, since other changes in incubation conditions which decreased the growth rate (lower temperature, low thymine levels) also resulted in lower specific activities of these enzymes. When the concentrations of folate present in the growth medium exceed 1.4 x 10-9 m, the specific activity of 10-formyl tetrahydrofolate synthetase and dihydrofolate reductase is decreased, possibly through repression of the enzyme. This effect, which may be of importance in the regulation of 1-carbon intermediates, was not observed for 5,10-methylene tetrahydrofolate dehydrogenase or for enzymes not involved in folate metabolism.

The recovery of liver tetrahydrofolate dehydrogenase activity from inhibition by methotrexate

Rat liver tetrahydrofolate dehydrogenase activity in vivo recovers rapidly from inhibition by methotrexate, despite the known stability of the enzyme-inhibitor complex. Control levels of activity are usually regained within 24 hr following a single injection of methotrexate. A similar pattern of recovery of activity has been found using the Guerin T8 epithelioma. Evidence that the enzyme is actually inhibited in situ has been obtained using a tetrazolium assay system. No evidence could be found for metabolism of the methotrexate to a form inactive with respect to tetrahydrofolate dehydrogenase. Synthesis of new enzyme with retention of the inhibited enzyme did not appear to play a major role in recovery of activity. Experiments using tritiated methotrexate indicated that both in vivo and in vitro, recovery of enzymic activity is probably associated with a dissociation of the enzyme-inhibitor complex.

A colorimetric assay system for tetrahydrofolate dehydrogenase

A colorimetric assay system for tetrahydrofolate dehydrogenase

Tetrahydrofolate dehydrogenase from bakers' yeast

Tetrahydrofolate dehydrogenase from bakers' yeast

FORMATION OF FOLATE ENZYMES DURING THE GROWTH CYCLE OF BACTERIA. 3. CHANGES IN TETRAHYDROFOLATE DEHYDROGENASE ACTIVITY DURING THE ACTIVE GROWTH PHASES OF STREPTOCOCCUS THERMOPHILUS AND LACTOBACILLUS ARABINOSUS.

FORMATION OF FOLATE ENZYMES DURING THE GROWTH CYCLE OF BACTERIA. 3. CHANGES IN TETRAHYDROFOLATE DEHYDROGENASE ACTIVITY DURING THE ACTIVE GROWTH PHASES OF STREPTOCOCCUS THERMOPHILUS AND LACTOBACILLUS ARABINOSUS.

Vitamin B12 and DNA Biogenesis

The enzyme, methylene tetrahydrofolic dehydrogenase, which mediates the reduction of one-carbon fragments from the formate to the formaldehyde level in the metabolic pathway leading to thymine methyl biogenesis, is reduced in activity in vitamin B12-deficient chick livers. The addition of vitamin or coenzyme B12 in vitro under aerobic conditions stimulates the deficient enzyme preparations with a Km of 2.9 × 10-10M. A test of the physiological significance of this stimulation has been made. Control and vitamin B12-deficient chickens were injected with tracer amounts of formate-C14 and formaldehyde-C14. With either tracer the specific activity of the purines and of thymine was diminished in the bone marrow of the deficient chicks, but the percentage decrease in the specific activity of thymine was greater than the percentage decrease in the purines. No difference was observed between the 2 tracers. It is concluded that a form of vitamin B12 plays a significant role in DNA synthesis, probably in the biogenesis of the thymine methyl.

Formation of Folate Enzymes during the Growth Cycle of Bacteria. I. Tetrahydrofolate Dehydrogenase Activity During the Lag and Acceleration Periods of Streptococcus faecalis R.

Formation of Folate Enzymes during the Growth Cycle of Bacteria. I. Tetrahydrofolate Dehydrogenase Activity During the Lag and Acceleration Periods of Streptococcus faecalis R.