Folate Polyglutamates Research Articles

Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme.

Folyl polyglutamate synthetase has been partially purified from mouse liver, and the general features of this enzyme have been characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration, and affinity chromatography on ATP-agarose and resulted in a 350-fold increase in specific activity with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP, and Mg2+ while partial reaction rates were observed in the absence of KCl or beta-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37 degrees C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a molecular weight of 65 000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate, and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. These data suggest that all of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo and that 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.

Folylpolyglutamate synthetase from beef liver: assay, stabilization, and characterization

A reliable assay for folylpolyglutamate synthetase has been devised and tested. Conditions have been established for the complete separation of [3H]glutamate and the tritium-labelled products on columns of DEAE-cellulose. The availability of this assay has aided us in partially purifying and characterizing the synthetase from extracts of beef liver. Suitable conditions have been found for the stabilization of the activity of both crude and partially purified folylpolyglutamate synthetase. The apparent Km values for L-glutamate (0.82 mM), dl-L-tetrahydrofolate (9 microM), ATP (25 microM with 10 mM MgCl2), KCl (3 mM), and 2-mercaptoethanol (5 mM) have been estimated. Several oxidized pteridine substrates have been tested. Of the antifolates tested, aminopterin is the more active substrate. The chain lengths of folate polyglutamates have been measured by chromatography on columns of DEAE-cellulose, with elution by a gradient of sodium acetate. Conjugates as long as hexaglutamates have been detected. The identities of the polyglutamates of tetrahydrofolate have been verified by hydrolysis in the presence of conjugase and by double-labelling experiments.

Hormonal alteration of methotrexate and folate polyglutamate formation in cultured hepatoma cells

Glutamylation of the antifolate methotrexate in H35 hepatoma cells was stimulated by physiologic concentrations of insulin and dexamethasone. At saturating concentrations of the hormone a 2.7-fold stimulation could be obtained with insulin (65 n m, 16-h exposure) and a 1.8-fold stimulation with dexamethasone (100 n m, 16-h exposure). The increases in glutamylation caused by the hormones were not additive, and both were inhibited by actinomycin D and cycloheximide. N 6, O 2′-dibutyryl cAMP and theophylline caused a modest reduction of glutamylation in control and dexamethasone-treated cultures, but repressed the stimulation caused by insulin by approximately one-third. Enhancement of synthesis by dexamethasone and insulin was associated with increases in the tri-, tetra-, and pentaglutamate derivatives of methotrexate, with little change in intracellular methotrexate and methotrexate diglutamate. When the conversion of folinic acid into the folylpolyglutamate pool was examined in folate-depleted H35 cells, insulin and dexamethasone had similar effects. The results suggest that these hormones play a role in the glutamylation of the folate coenzymes in a liver-derived transformed cell line in culture and that these effects are also reflected in the interaction of the cells with antifolates such as methotrexate.

Serum cobalamin concentration in sickle cell disease (HbSS).

The serum cobalamin, serum folate, erythrocyte folate concentrations and total leucocyte and neutrophil counts have been studied in sickle cell children (with HbSS) during the steady state. There was a significant reduction in serum cobalamin concentration with significant increases in both the total leucocyte count and neutrophil differential. A relationship was also observed between the serum cobalamin and erythrocyte folate. The lowest erythrocyte folate levels were observed in patients with the lowest serum cobalamin concentration. These abnormalities are discussed in relation to the possible role of cobalamin in the synthesis of erythrocyte folate polyglutamate and possible immunological role of cobalamin among these children. These results lend support to the view that daily cobalamin supplementation along with folate (which is being given) may prove beneficial to the sickle cell patients as it may lower the frequency and severity of infections.

Methotrexate analogues. 20. Replacement of glutamate by longer-chain amino diacids: effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activity.

Chain-extended analogues of methotrexate were synthesized by condensation of 4-amino-4-deoxy-N10-methylpteroic acid with esters of L-alpha-aminoadipic, L-alpha-aminopimelic, and L-alpha-aminosuberic acids, followed by ester hydrolysis with acid or base. Coupling was accomplished in up to 85% yield by the use of the peptide bond forming reagent diethyl phosphorocyanidate at room temperature. The products were found to bind bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase with an affinity comparable to methotrexate and were also equitoxic to L1210 cells in culture. Cytotoxicity increased up to 3-fold as the number of CH2 groups in the amino acid side chain was extended from two to five. The alpha-aminoadipate and alpha-aminopimelate analogues were poor substrates for carboxypeptidase G1, confirming that this enzyme has a strict requirement for a C-terminal L-glutamic acid residue. The in vivo antitumor activity of the chain-extended analogues against L1210 leukemia in mice was comparable to that of the parent drug on the qd X 9 schedule, but higher doses were required to achieve the same increase in survival. The results were consistent with findings, reported separately, that these compounds are poor substrates for folate polyglutamate synthetase and therefore would not be expected to form gamma-polyglutamates once they enter a cell. This distinctive property has potential therapeutic implications for the treatment of certain MTX-resistant tumors whose resistance may be associated with a lower than normal capacity to form gamma-polyglutamates in comparison with proliferative tissues such as intestinal mucosa or marrow.

Effects of phenobarbitone and phenytoin on folate catabolism in the rat

The effects of phenobarbitone and phenytoin on the catabolism of oral [2- 14C] and [3′,5′,7,9- 3H] folic acid were investigated. Normal rats were found to excrete an excess of 3H-labelled compounds into the urine and 14C-labelled compounds into the faeces. Phenytoin abolished this urinary 3H imbalance and also delayed and prolonged the overall excretion of radioactive material. Phenobarbitone appeared to increase the amounts of urinary scission products in the first 24 hr but over the 0–72 hr period both anticonvulsants decreased folate polyglutamate catabolism. As the anticonvulsants used in these experiments decreased folate catabolism in the rat it is unlikely that the megaloblastic anaemia caused by chronic anticonvulsant therapy is due to induction of the enzymes responsible for folate breakdown in vivo.

A critical intracellular concentration of fully reduced non-methylated folate polyglutamates prevents macrocytosis and diminished growth rate of human cell line K562 in culture.

Growth rate of human leukaemic cell line K562 was independent of intracellular folate concentration when this was greater than 1.5 microM. When intracellular folate concentration was less than 1.5 microM, the rate of growth was proportional to the logarithm of intracellular concentration of non-methylated fully reduced folates, but not to the logarithm of the intracellular concentration of N5-methyltetrahydropteroylglutamate. Intracellular folate concentration sufficient to support an optimal growth rate was maintained by either DL-N5-formyltetrahydropteroylglutamate or DL-N5-methyltetrahydropteroylglutamate at a 100-fold lower concentration than pteroylglutamate. Addition of hypoxanthine to culture medium partially restored growth of folate-depleted cells: thymidine had no effect on growth rate either alone or in combination with thymidine. Folate-depleted cells with diminished growth rate were larger than replete cells, but did not have megaloblastic morphology. The mitotic index was not decreased in cultures with diminished growth rate. The rate of growth and cell size of K562 cells is thus dependent on a critical intracellular concentration of non-methylated tetrahydrofolates, which may be maintained by different concentrations of either reduced folates or pteroylglutamate.

Chronic cobalamin inactivation impairs folate polyglutamate synthesis in the rat.

Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.

Characterization of the function of mammalian folylpolyglutamate synthetase (FPGS).

The function and characteristics of mouse folylpolyglutamate synthetase have been examined. Folate polyglutamates were poor substrates for the efflux mechanism for monoglutamates in L1210 mouse leukemia cells, with negligible loss of preformed folate polyglutamates to the medium over four hours. Disruption of folate metabolism with methotrexate did not augment efflux of folate polyglutamates. Folylpolyglutamate synthetase, partially purified from mouse liver, was found to accept a variety of folate derivatives as substrates, including pteroic acid and methotrexate; however, the concentration of these substrates that saturated the reaction varied considerably. The enzyme that catalyzed the addition of glutamic acid to methotrexate and to the naturally-occurring folate monoglutamates appeared to be the same. The cytotoxicity of folylpolyglutamate synthetase inhibitors was predicted to require continued cell division since their effects would be based upon a decreased rate of synthesis of folate cofactors capable of retention by the cell membrane. Hence folylpolyglutamate synthetase inhibitors should have low toxicity to nonproliferative cell populations.

Folate oligoglutamate:amino acid transpeptidase.

A new and previously unrecognized enzyme activity folate oligoglutamate:amino acid transpeptidase (folate transpeptidase), was detected in rat liver extracts. This activity involves the exchange of the terminal gamma-glutamyl residue (where PteGlu represents pteroylglutamic acid) of H4PteGlu-gamma-Glu, PteGlu-gamma-Glu, or p-aminobenzoyl-Glu-gamma-Glu for an amino acid such as [14C]glutamic acid, [14C]methionine, [14C]glutamine, or [14C]glycine. In the case of substrates PteGlu2 and [14C]Glu, the product is PteGlu-gamma-[14C]Glu. The enzyme responsible for catalyzing this activity was purified 39,000-fold by precipitation of inactive protein, fractionation on anionic and molecular sieve columns, and chromatography on diaminohexane-Sepharose. The enzyme functions maximally at pH 7.9, shows an absolute requirement for sulfhydryl reductants, and appears to have a molecular weight of 65,000. The function of folate transpeptidase is not known. A second, though much weaker, enzyme activity, tetrahydrofolic acid:amino acid ligase (folate ligase), co-purified with folate transpeptidase through all steps. Folate transpeptidase and the associated folate ligase activity were separated from folate polyglutamate synthetase during the initial steps of the purification procedure.

Purification and partial characterization of rat liver folate binding protein: cytosol I.

The high molecular weight folate binding protein of rat liver cytosol has been purified to apparent homogeneity. Purification was achieved by using a combination of gel filtration, O-(diethylaminoethyl)cellulose chromatography, and affinity chromatography. This folate binding protein was initially identified during purification by an in vivo labeling procedure involving intraperitoneal injection of [3H]folic acid prior to sacrifice and subsequently by its ability to bind naturally reduced [3H]folate polyglutamates in vitro. A molecular weight of 210 000 was estimated by gel chromatography. This is distinct from the trifunctional formyl-methenyl-methylene synthetase of rat liver which has a molecular weight of 225 000. Sodium dodecyl sulfate electrophoresis revealed a single band with a molecular weight of about 100 000 which suggests the native protein is composed of two identical subunits. The partially purified protein contains bound tetrahydropteroylpentaglutamate.

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

Folate pentaglutamate and folate hexaglutamate mediated one-carbon metabolism.

The proposal that folate polyglutamate cofactors of different chain lengths function differently in metabolism was investigated. We identified the one-carbon units present in rat liver folates within each of the liver folate polyglutamate groups, the folate penta-, hexa-, and heptaglutamates. This identification revealed that at the pentaglutamate level, 5-methyl-H4folate was the major form, accounting for 18% of the total liver folates, with small amounts of H4folate. At the hexaglutamate level, 5-methyl-H4folate and H4folate were major forms, accounting for 17% and 22% of the liver folates, respectively. At the heptaglutamate level, 5-methyl-H4folate occurred in small amounts while H4folate predominated. The rats used here had been fed a diet low in methionine and were exposed to N2O gas. These results are qualitatively similar to those from rat brain [Brody, T., Shin, Y. S., & Stokstad, E. L. R. (1976) J. Neurochem. 27, 409--413] where 5-methyl-H4folate was a major fraction of the folate pentaglutamates but a minor, if detectable, part of the hexa- and heptaglutamates. The folates 5-methyl-H4PteGlu5-7 were metabolically active in the liver, as illustrated by the severe contraction in the amounts of these folates following an injection of methionine. This indicates that "folate-binding proteins" do not prevent 5-methyl-H4folates from entering into one-carbon metabolism.

The effects of nitrous oxide on cobalamins, folates, and on related events.

The anaesthetic gas nitrous oxide (N2O), when inhaled for longer than 6 hr, produces megaloblastic anemia in man. Longer term inhalation, as in addicts, produces a syndrome similar to that due to B12 neuropathy, and long term exposure to low concentrations results in an increased abortion rate and neuropathy, particularly in dental personnel. N2O acts by oxidizing vitamin B12 from the active reduced cob[I]alamin form to the inactive cob[III]alamin form. In turn, this inactivates the enzyme methionine synthetase which requires both B12 and folate as cofactors. In the rat, hepatic methionine synthetase is completely inactivated after 3 hr exposure to a mixture of equal parts of N2O/O2. There is an impared uptake of folate analogues by the liver so that the plasma folate level rises and thereafter there is a considerable loss of folate into the urine. Hepatic folate concentration falls to 25% within 10 days of N2O exposure. There is a failure to synthesize folate polyglutamate (the active folate coenzyme) from all other than formyltetrahydrofolate. As oxidization of the methyl of methionine is an important source of formyl, the failure of methionine synthesis in turn appears to lead to the failure in supply of formate and, hence, a lack of the formylfolate substrate.

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.

Abnormal biochemistry and morphology of erythrocytes in a case of transcobalamin II deficiency during treatment

AbstractMetabolic studies on a transcobalamin II deficient child during apparent haematological remission revealed abnormally low erythrocyte levels of cobalamin‐ and folate‐coenzymes and highly unusual morphology of erythrocytes. Transfusion with hydroxocobalamin‐supplemented plasma temporarily normalized the child's erythrocyte adenosylcobalamin and methylcobalamin. Subsequent oral leucovorin increased erythrocyte levels of low‐polyglutamate folate. These findings provide further evidence that TCII may be involved not only in cellular cobalamin uptake but also in intracellular conversion of the cobalamin coenzymes and polyglutamate folates.

Changes in tissue folates accompanying nitrous oxide-induced inactivation of vitamin B12 in the rat

The anesthetic gas, nitrous oxide, oxidizes cob(I)alamin and thus inactivates methionine synthetase which requires cobalamin as a coenzyme. The effect on folates in liver, kidney, marrow, plasma, and brain in rats breathing a 1/1 nitrous oxide/oxygen mixture is described. There is loss of folate from tissues, most marked in liver, that affects folate polyglutamates to a greater extent than folate monoglutamates. Both methyl- and nonmethyl-analogues are affected. There is a transient rise in the levels of 5-methyltetrahydropteroylpolyglutamate in all tissues 8 h after starting nitrous oxide, which falls thereafter. In marrow and brain there is also a transient rise in methyltetrahydropteroylmonoglutamate. Plasma folate increased markedly throughout the period of exposure to nitrous oxide. It is suggested that these changes are due to the action of nitrous oxide in depressing tissue uptake of folate from plasma, in promoting loss of folate into urine and in inhibiting folate polyglutamate synthesis.

Properties of Folic Acid γ-Glutamyl Hydrolase (Conjugase) in Rat Bile and Plasma

The properties of folic acid γ-glutamyl hydrolase (conjugase) [EC 3.4.12.10] in rat bile and plasma were investigated. Conjugase activity in bile showed two pH optima; one at pH 4.5–5.0 and one at pH 6.7–7.5. The enzyme activity in plasma had a broad pH—profile with an optimum at pH 6.2–7.5. Conjugase activity from both bile and plasma was inhibited in the presence of the sulfhydryl reagent, p-hydroxymercuriphenylsulfonate, and stimulated in the presence of 2-mercaptoethanol. Conjugase activity in bile was inhibited by Zn2+ at pH 7.5 but not at pH 4.5 and was much more stable to heat at pH 4.5. No separation of the biliary conjugase activity measured at the two different pH values was obtained by Sephadex G-150 chromatography. Secretion of biliary conjugase was essentially constant over a 6-hour period when activity was assayed at pH 4.5 or pH 7.5. The enzyme in bile converted pteroyltriglutamate to a mixture of about 5% glutamate and 95% γ-glutamylglutamate at either pH, whereas the enzyme in plasma produced 23% glutamate and 77% γ-glutamylglutamate. The possible contribution of biliary conjugase to intestinal absorption of folate polyglutamates is discussed.folic acid γ-glutamyl hydrolase bile

The effect of nitrous oxide-induced vitamin B 12 deficiency on [formula omitted] folate metabolism

The effect of N 2O-induced vitamin B 12 deficiency on in vivo folate metabolism was studied in an animal model previously developed for studies of the folate enterohepatic cycle, and in rats with localized, subcutaneous tumor nodules. While N 2O inhibited liver folate polyglutamate formation, it did not affect the absorption of ( 3H)PteGlu 1 from the gut, its reduction, methylation, and transport to the liver, or the subsequent secretion of CH 3H 4( 3H)PteGlu 1 into bile—the folate enterohepatic cyle. In addition, N 2O did not impair folate polyglutamate formation in the fibrosarcoma tumor nodule suggesting that tumor tissue can either demethylate CH 3H 4PteGlu 1 by an alternate pathway or can utilize it as a substrate for polyglutamate formation without demethylation.

An inverse relationship of rat liver folate polyglutamate chain length to nutritional folate sufficiency

The relative concentrations of folylpolyglutamates of differing chain length in rat liver and the uptake of exogenous [ 3H]folic acid (20 μCi, 20 μg) into liver folylpolyglutamates were examined in rats maintained on (a) standard and folate-supplemented standard diets and (b) semi-defined folate-sufficient and folate-deficient diets. Folylpolyglutamates extracted from liver were cleaved to p-aminobenzolpolyglutamates which were separated by ion-exchange chromatography. The relative concentrations and ultimate radiolabeling of longer-chain folylpolyglutamates (six, seven and eight glutamate residues) were greatest in the livers of folate-deficient rats, whereas the intermediate-chain folylpolyglutamates (three, four and five flutamate residues) were the greatest portion of total liver folates of folate-supplemented rats. Thus, the length of the polyglutamate chain added to liver folates is inversely related to the total concentration of liver folates. These data suggest that folylpolyglutamate biosynthesis in the liver may be controlled by the liver folate concentrations. In folate insufficiency such a control mechanism would serve to enhance the affinity of folates for folate-dependent enzymes and to conserve the liver folate concentration.