Formyltetrahydrofolate synthetase. The role of the sulfhydryl groups.
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Adenosine Triphosphate↗
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Cation-dependent reassociation of subunits of N10-formyltetrahydrofolate synthetase from Clostridium acidi-urici and Clostridium cylindrosporum.
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Adenine Nucleotides↗
Formyltetrahydrofolate synthetase. A study of equilibrium reaction rates.
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Adenine Nucleotides↗
Formyltetrahydrofolate synthetase. Initial velocity and product inhibition studies.
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Adenosine Triphosphate↗
Formyltetrahydrofolate synthetase. Magnetic resonance studies of the reaction.
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Adenine Nucleotides↗
l-5-formyltetrahydrofolate and l-5-methyltetrahydrofolate rescue in L1210 leukemia treated with high methotrexate doses.
Highly purified 5-l-methyltetrahydrofolate (m-THF) and 5-l-formyl-THF (f-THF) preparations were compared for rescuing from methotrexate (MTX) toxicity in DBA2 mice transplanted with L1210 leukemia. Mice received two doses of reduced folates (2 mg/kg, s.c.) 16 and 24 h after a single s.c. MTX dose. f-THF was 1.8 time more effective than m-THF in protecting tumor cells from MTX (800 mg/kg). This MTX dose caused a 57% fall in circulating polymorphonucleates, which was prevented by both reduced folates. Treatment with 800 mg/kg of MTX plus m-THF was 1.5 fold more effective than the same MTX dose plus f-THF in increasing survival time of tumor-bearing mice. These data suggest a higher selectivity and efficacy of l-m-THF with respect to l-f-THF in rescuing from MTX toxicity.
Animals↗
The regulation of folate and methionine metabolism.
1. The isolated perfused rat liver and suspensions of isolated rat hepatocytes fail to form glucose from histidine, in contrast with the liver in vivo. Both rat liver preparations readily metabolize histidine. The main end product is N-formiminoglutamate. In this respect the liver preparations behave like the liver of cobalamin- or folate-deficient mammals. 2. Additions of L-methionine in physiological concentrations (or of ethionine [2-amino-4-(ethylthio)butyric acid]) promotes the degradation of formiminoglutamate, as is already known to be the case in cobalamin of folate deficiency. Added methionine also promotes glucose formation from histidine. 3. Addition of methionine accelerates the oxidation of formate to bicarbonate by hepatocytes. 4. A feature common to cobalamin-deficient liver and the isolated liver preparations is taken to be a low tissue methionine concentration, to be expected in cobalamin deficiency through a decreased synthesis of methionine and caused in liver preparations by a washing out of amino acids during the handling of the tissue. 5. The available evidence is in accordance with the assumption that methionine does not directly increase the catalytic capacity of formyltetrahydrofolate dehydrogenase; rather, that an increased methionine concentration raises the concentration of S-adenosylmethionine, thus leading to the inhibition of methylenetetrahydrofolate reductase activity [Kutzbach & Stokstad (1967) Biochim. Biophys. Acta 139, 217-220; Kutzbach & Stokstad (1971) Methods Enzymol. 18B, 793-798], that this inhibition causes an increase in the concentration of methylenetetrahydrofolate and the C1 tetrahydrofolate derivatives in equilibrium with methylenetetrahydrofolate, including 10-formyltetrahydrofolate; that the increased concentration of the latter accelerates the formyltetrahydrofolate dehydrogenase reaction, because the normal concentration of the substrate is far below the Km value of the enzyme for the substrate. 6. The findings are relevant to the understanding of the regulation of both folate and methionine metabolism. When the methionine concentration is low, C1 units are preserved by the decreased activity of formyltetrahydrofolate dehydrogenase and are utilized for the synthesis of methionine, purines and pyrimidines. On the other hand when the concentration of methionine, and hence adenosylmethionine, is high and there is a surplus of C1 units as a result of excess of dietary supply, formyltetrahydrofolate dehydrogenase disposes of the excess. When ample dietary supply causes an excess of methionine, which has to be disposed of by degradation, the increased activity of formyltetrahydrofolate dehydrogenase decreases the supply of methyltetrahydrofolate. Thus homocysteine, instead of being remethylated, enters the pathway of degradation via cystathionine. 7. The findings throw light on the biochemical abnormalities associated with cobalamin deficiency (megaloblastic anaemia), especially on the 'methylfolate-trap hypothesis'. This is discussed. 8...
Amino Acids↗
Assembly of an active enzyme by the linkage of two protein modules.
The feasibility of creating new enzyme activities from enzymes of known function has precedence in view of protein evolution based on the concepts of molecular recruitment and exon shuffling. The enzymes encoded by the Escherichia coli genes purU and purN, N10-formyltetrahydrofolate hydrolase and glycinamide ribonucleotide (GAR) transformylase, respectively, catalyze similiar yet distinct reactions. N10-formyltetrahydrofolate hydrolase uses water to cleave N10-formyltetrahydrofolate into tetrahydrofolate and formate, whereas GAR transformylase catalyses the transfer of formyl from N10-formyltetrahydrofolate to GAR to yield formyl-GAR and tetrahydrofolate. The two enzymes show significant homology (approximately 60%) in the carboxyl-terminal region which, from the GAR transformylase crystal structure and labeling studies, is known to be the site of N10-formyltetrahydrofolate binding. Hybrid proteins were created by joining varying length segments of the N-terminal region of the PurN gene (GAR binding region) and the C-terminal (N10-formyltetrahydrofolate binding) region of PurU. Active PurN/PurU hybrids were then selected for by their ability to complement an auxotrophic E. coli strain. Hybrids able to complement the auxotrophs were purified to homogeneity and assayed for activity. The specific activity of two hybrid proteins was within 100- to 1000-fold of the native purN GAR transformylase validating the approach of constructing an enzyme active site from functional parts of others.
Acyltransferases↗
C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains.
C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
Aminohydrolases↗
Analysis and biochemistry of blood folate.
Although the analysis of low plasma concentrations of 5-methyltetrahydrofolate by several specific HPLC methods has been reported, considerably fewer routine chromatographic techniques exist for the analysis of specific folate coenzymes in the erythrocyte where a nonspecific bioassay indicates that the vitamin achieves a level 10 times higher than that in plasma. By using three separate folypolyglutamate deconjugation procedures and combining an extraction technique which adequately preserves all native folate coenzymes with an HPLC technique utilizing fluorescence, diode array, and off-line radioassay detection capable of resolving all crucial native folates in their monoglutamyl forms, we were unable to demonstrate levels of 5-methyltetrahydrofolate in whole blood hemolysate beyond what might be expected from the plasma component. While the exact nature of erythrocyte folate could not be ascertained, we provide evidence that a proportion of it may exist at the formyl level of oxidation. The complex pH and enzymatic interrelationship between folate coenzymes at the formyl oxidation level is discussed in terms of our extraction technique and findings, as well as in a broader biological context. This paper also describes a simple acid precipitation technique for measuring plasma 5-methyltetrahydrofolate, as well as providing comprehensive data on the chromatographic behavior of all the folylmonoglutamates in reversed-phase and weak anion-exchange modes, including useful spectral data for optimizing detection parameters and identifying individual coenzymes. 10-Formyltetrahydrofolate and 5-methyltetrahydrofolate are the two most important one-carbon-substituted folate coenzymes. 10-Formyltetrahydrofolate is unavailable commercially, probably due to its instability. We chart the chemical synthesis of this important coenzyme and show that it and what is thought to be 5,10-hydroxymethylenetetrahydrofolate are actually minor products compared to the parent 5,10-methenyltetrahydrofolate and the ultimate reaction product, 5-formyltetrahydrofolate. Since intraerythrocyte folate binds to a specific hemoglobin site, we ascertained the total number of binding sites on hemoglobin (Bmax) and the equilibrium dissociation constant (Kd) for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and the antimetabolite methotrexate. Binding affinities were consistent with a low-affinity, low-capacity interaction for all three. It was demonstrated that hemoglobin has a greater affinity for 5-methyltetrahydrofolate than for the other folate derivatives (Kd = 1.2 x 10(-3) M), while rather surprisingly, methotrexate had a higher affinity for hemoglobin than did 5-formyltetrahydrofolate (Kd = 2.5 x 10(-3) and 3.7 x 10(-2) M, respectively.
Binding Sites↗
Intracellular metabolism of 5-formyl tetrahydrofolate in human breast and colon cell lines.
This report describes the intracellular metabolism of 5-formyltetrahydrofolate into the various one-carbon substituted folate and polyglutamate pools in a human breast (MCF-7) and colon (HCT 116) carcinoma cell line. Metabolism into the one-carbon substituted pools was found to be time and dose dependent over a concentration range up to 50 microM. A 3-fold increase in total intracellular folate was noted over a 50-fold concentration range (1-50 microM) of 5-formyltetrahydrofolate tested in the colon cell line, while in the breast line, a 6-fold increase was detected over a 500-fold concentration range (0.1-50 microM). The level of 5, 10-methylenetetrahydrofolate, which was detectable only in the breast cell line, was found to increase by a factor of 10 (1.8 pmol/mg to 17.9 pmol/mg) over the concentration range studied. The majority of metabolism was into the 10-formyltetrahydrofolate and tetrahydrofolate pools in the breast cells and into the 5-methyltetrahydrofolate pool in the colon cells. Polyglutamation was also time and dose dependent, with a significant proportion of the total pool represented by the higher polyglutamate forms (Glu3-Glu5) after 24 h of continuous exposure to 5-formyl tetrahydrofolate. Pentaglutamate was the highest level noted in both cell lines. The intracellular half-life of the polyglutamate forms was inversely related to the length of the polyglutamate tail with half-lives of 71, 131, 143, 441, and 1167 min for the mono- through pentaglutamate, respectively. Finally, up to a 20:1 ratio of the biologically inactive (6R) isomer to active (6S) isomer of 5-formyltetrahydrofolate resulted in no effect on metabolism into the one-carbon substituted folate pools and only minimal decreases in metabolism to the polyglutamate forms. These studies suggest that prolonged exposure to even relatively low doses of 5-formyltetrahydrofolate may be optimal for intracellular metabolism to the most biologically relevant forms for ternary complex formation with thymidylate synthase and fluorodeoxyuradylate, since longer exposures result in a greater accumulation of the higher polyglutamates.
Breast Neoplasms↗
Role for cytosolic folate-binding proteins in the compartmentation of endogenous tetrahydrofolates and the 5-formyl tetrahydrofolate-mediated enhancement of 5-fluoro-2'-deoxyuridine antitumor activity in vitro.
A major portion of the intracellular folates in L1210 cells grown in (6R,S)-5-formyltetrahydrofolate (leucovorin) was bound to cytosolic proteins when cell extracts were fractionated by rapid gel filtration or adsorption with activated charcoal. Only low levels of intracellular folates were associated with mitochondria (less than 5%). Protein-bound folates comprised 37-100% of the cytosolic cofactors following growth in 2-600 nM 5-formyltetrahydrofolate. Total intracellular folates increased in proportion to the changes in media folate concentration; however, binding was saturable. The maximum level of protein-bound folates in L1210 cells was 66 pmol/mg protein. Protein-bound folates were also detected in HT29 human colon adenocarcinoma cells grown in 5-formyltetrahydrofolate (maximum, 11 pmol/mg protein). For both lines, folate binding was specific for the tetrahydrofolate and 5,10-methylenetetrahydrofolate pool, and, to a lesser extent, 5-methyltetrahydrofolate. Extremely low levels of protein-bound 5-formyl-, 10-formyl-, and 5,10-methenyltetrahydrofolates were measured, even though considerable amounts were detected intracellularly. Pentaglutamyl folates were the predominant cofactor forms in L1210 cells; conversely, the tetraglutamates were the most abundant protein-bound folate derivatives. Increasing media concentrations of 5-formyltetrahydrofolate potentiated 5-fluoro-2'-deoxyuridine cytotoxicity. For L1210 cells, essentially all of the intracellular tetrahydrofolate and 5,10-methylenetetrahydrofolate fraction was protein bound over the concentration range of 5-formyltetrahydrofolate which maximally augmented fluoropyrimidine cytotoxicity. The relative changes in the 50% inhibitory concentrations for fluorodeoxyuridine directly approximated the increases in the levels of protein-bound tetrahydrofolates in L1210 cells. There was no direct relationship between the levels of unbound folates and fluorodeoxyuridine cytotoxicity. Similar results were obtained with HT29 cells. The major folate-binding protein in L1210 cells eluted during Sephacryl S-300 chromatography with a molecular weight of approximately 200,000; a small amount of a higher molecular weight folate-binding protein (Mr 450,000) was also detected. These findings support the concept of a compartmentation of endogenous folates involving specific binding to cytosolic proteins. These associations may regulate reduced folate availability for metabolic processes, and also mediate utilization of 5,10-methylenetetrahydrofolate for ternary complex formation with thymidylate synthase in cells treated with fluoropyrimidines. In this fashion, the levels of protein-bound tetrahydrofolates could represent an additional, previously unrecognized, determinant of fluoropyrimidine pharmacological activity toward mammalian cells.
Adenocarcinoma↗
Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates.
This study reports the isolation and characterization of a variant of the human CCRF-CEM leukemia cell line that overproduces the carrier protein responsible for the uptake of reduced folates and the folate analogue methotrexate. The variant was obtained by adapting CCRF-CEM cells for prolonged times to stepwise decreasing concentrations of 5-formyltetrahydrofolate as the sole folate source in the cell culture medium. From cells that were grown on less than 1 nM 5-formyl-tetrahydrofolate, a variant (CEM-7A) was isolated exhibiting a 95-fold increased Vmax for [3H]methotrexate influx compared to parental CCRF-CEM cells. The values for influx Km, efflux t0.5, and Ki for inhibition by other folate (analogue) compounds were unchanged. Affinity labeling of the carrier with an N-hydroxysuccinimide ester of [3H]methotrexate demonstrate an approximately 30-fold increased incorporation of [3H] methotrexate in CEM-7A cells. This suggests that the up-regulation of [3H]methotrexate influx is not only due to an increased amount of carrier protein, but also to an increased rate of carrier translocation or an improved cooperativity between carrier protein molecules. Incubation for 1 h at 37 degrees C of CEM-7A cells with a concentration of 5-formyltetrahydrofolate or 5-methyltetrahydrofolate in the physiological range (25 nM) resulted in a 7-fold decline in [3H]methotrexate influx. This down-regulation during incubations with 5-formyltetrahydrofolate or 5-methyltetrahydrofolate could be prevented by either the addition of 10-25 nM of the lipophilic antifolate trimetrexate or by preincubating CEM-7A cells with 25 nM methotrexate. The down-regulatory effect was specifically induced by reduced folates since incubation of CEM-7A cells with 25 nM of either methotrexate, 10-ethyl-10-deazaaminopterin, aminopterin, or folic acid, or a mixture of purines and thymidine, had no effect on [3H]methotrexate influx. Similarly, these down-regulatory effects on [3H]methotrexate transport by 5-formyltetrahydrofolate, and its reversal by trimetrexate or methotrexate, were also observed, though to a lower extent, for parental CCRF-CEM cells grown in folate-depleted medium rather than in standard medium containing high folate concentrations. These results indicate that mediation of reduced folate/methotrexate transport can occur at reduced folate concentrations in the physiological range, and suggest that the intracellular folate content may be a critical determinant in the regulation of methotrexate transport.
Biological Transport↗
Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance.
Gram-negative bacteria have evolved mechanisms to resist the bactericidal action of cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. The strategy involves the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A in their outer membrane. ArnA is a key enzyme in the Ara4N-lipid A modification pathway. It is a bifunctional enzyme catalyzing (1) the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) to the UDP-4' '-ketopentose [UDP-beta-(l-threo-pentapyranosyl-4' '-ulose] and (2) the N-10-formyltetrahydrofolate-dependent formylation of UDP-Ara4N. Here we demonstrate that the transformylase activity of the Escherichia coli ArnA is contained in its 300 N-terminal residues. We designate it the ArnA transformylase domain and describe its crystal structure solved to 1.7 A resolution. The enzyme adopts a bilobal structure with an N-terminal Rossmann fold domain containing the N-10-formyltetrahydrofolate binding site and a C-terminal subdomain resembling an OB fold. Sequence and structure conservation around the active site of ArnA transformylase and other N-10-formyltetrahydrofolate-utilizing enzymes suggests that the HxSLLPxxxG motif can be used to identify enzymes that belong to this family. Binding of an N-10-formyltetrahydrofolate analogue was modeled into the structure of ArnA based on its similarity with glycinamide ribonucleotide formyltransferase. We also propose a mechanism for the transformylation reaction catalyzed by ArnA involving residues N(102), H(104), and D(140). Supporting this hypothesis, point mutation of any of these residues abolishes activity.
Amino Acid Sequence↗
Compartmentation of folate metabolism in rat pancreas: nitrous oxide inactivation of methionine synthase leads to accumulation of 5-methyltetrahydrofolate in cytosol.
Folate-dependent one-carbon metabolism and methylation reactions have been implicated in the secretory function of the pancreas. Because vitamin B-12 deficiency perturbs folate metabolism, we determined the effects of nitrous oxide inactivation of methionine synthase on the compartmentation of folate metabolism in rat pancreas. Rats were exposed to an atmosphere of nitrous oxide and oxygen (80 and 20%, respectively) for 18 h; control rats breathed air. Folate coenzyme concentrations were determined by HPLC and Lactobacillus casei microbiological assay of the cytosolic and mitochondrial fractions of pancreas, which contained 62 and 46%, respectively, of the total folate. In pancreas of control rats, cytosolic folates were 5-methyltetrahydrofolate (31% of total folates), tetrahydrofolate (54%) and 5- and 10-formyltetrahydrofolate (6 and 8%, respectively). In the rats exposed to nitrous oxide, cytosolic 5-methyltetrahydrofolate concentrations were significantly greater (59% of total folates) and tetrahydrofolate concentrations were significantly lower (32%) than in controls; however, total cytosolic folate levels were unaffected by nitrous oxide exposure. In controls, mitochondrial folates were composed of 5-methyltetrahydrofolate (9% of total folates), tetrahydrofolate (60%) and 5- and 10-formyltetrahydrofolate (22 and 10%, respectively). Exposure to nitrous oxide led to significantly lower total mitochondrial folates (1.49 +/- 0.18 vs. 0.75 +/- 0.29 nmol/g, control vs. nitrous oxide, P < 0.05). This was due to a significantly lower concentration of tetrahydrofolate and 5-formyltetrahydrofolate, but not of 5-methyl- or 10-formyltetrahydrofolate. The activity of methionine synthase was 85% lower (P < 0.001) in pancreatic extracts of rats exposed to nitrous oxide than in controls. These results show that cytosolic folates accumulate in pancreas as the 5-methyl derivative at the expense of other reduced folates, as happens in liver. However, in contrast to results in liver, the mitochondrial folate concentration was lower in the pancreas of rats exposed to nitrous oxide, and this decline was limited to the 5-formyl- and tetrahydrofolate derivatives.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Identification of a highly glycosylated methotrexate membrane carrier in K562 human erythroleukemia cells up-regulated for tetrahydrofolate cofactor and methotrexate transport.
A K562 human erythroleukemia line (designated K562.4CF) was selected for increased tetrahydrofolate cofactor transport in a growth-limiting concentration (0.4 nM) of (6R,S)-5-formyltetrahydrofolate. K562.4CF cells exhibited elevated methotrexate uptake relative to parental cells, attributable to a 10-fold increased influx Vmax. The rate of methotrexate efflux in K562.4CF cells was somewhat increased (55%) as well. The transport system in K562.4CF cells had similar and high apparent binding affinities for methotrexate and 5-formyltetrahydrofolate and a markedly reduced affinity for folic acid, properties typically associated with the "classical" methotrexate/tetrahydrofolate cofactor transporter in tumor cells. Methotrexate uptake in K562.4CF cells decreased substantially under nonselective conditions; high levels of transport were restored in 0.4 nM 5-formyltetrahydrofolate. Treatment of parental and K562.4CF cells with N-hydroxysuccinimide methotrexate inhibited methotrexate influx. N-Hydroxysuccinimide-[3H]methotrexate (700 nM) radiolabeled a broadly migrating band at Mr 76,000-85,000. Incorporation from N-hydroxysuccinimide-[3H]methotrexate into this band was increased 7-fold in K562.4CF over parental cells and was blocked by unlabeled methotrexate, (6S)-5-formyltetrahydrofolate, or, to a lesser extent, folic acid. Whereas incubation with endoglycosidase F had no effect on the electrophoretic migration of the labeled protein, treatment with endoglycosidase F and glycopeptidase F, or endo-beta-galactosidase, reduced the apparent molecular weight to Mr approximately 52,000 or approximately 58,000, respectively. These results suggest that the high-affinity transporter in K562.4CF cells is an N-linked glycoprotein containing internal beta-galactosidic linkages in, or immediately after, unbranched poly-N-acetyllactosamine sequences. Differences in the level of glycosylation may, in part, account for the disparity in the apparent sizes of the homologous folate transport proteins from human and murine cells.
Affinity Labels↗
Characteristics of a novel transport system for folate compounds in wild-type and methotrexate-resistant L1210 cells.
The growth requirements and transport characteristics of folate compounds in a methotrexate-resistant L1210/R81 cell line were compared with parental cells. Concentrations for half-maximal growth of the resistant cells with folate (350 nM) and 5-formyltetrahydrofolate (20 nM) were found to be higher by 2.7-fold and 20-fold, respectively, relative to the parent, suggesting that changes had occurred in the transport of these folate compounds. Transport measurements revealed that the resistant cells have lost the capacity to transport methotrexate and other folate compounds via the reduced-folate transport system but that a second previously undescribed transport system is present. Uptake of folate via this second route is energy dependent, exhibits saturation kinetics, can be inhibited by substrate analogues, and is activated by a reduction in pH. The pH effect is substantial since uptake at 5.0 microM folate can be increased 10-fold by decreasing the pH from 7.4 to 6.2. The observed Kt for half-maximal influx of folate at pH 6.8 was 5.2 microM, and the Vmax was 0.55 pmol/min/mg of protein. Ki values for methotrexate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were 10.2, 3.2, and 2.7 microM, respectively. Transport activity was not affected by depleting internal stores of folate by growth in folate-free medium, and this same transport system was also present in comparable amounts in wild-type cells. In the latter case, interfering uptake via the reduced-folate transport system was blocked by bromosulfophthalein and thiamine pyrophosphate. Methotrexate and 5-formyltetrahydrofolate were also transported via this alternative, pH-dependent route, whereas little or no uptake of 5-methyltetrahydrofolate could be detected. The results indicate that antifolate resistance in L1210/R81 cells was induced by inactivating the reduced-folate transport system and by utilizating a secondary route which has a low capacity for transporting methotrexate but whose activity is sufficient to permit cell growth in medium supplemented with folate or 5-formyltetrahydrofolate.
Animals↗
The role of folate deficiency in the development of peripheral neuropathy caused by anticonvulsants.
To investigate the role of folate deficiency in neuropathy caused by anticonvulsants, electrophysiological studies of peripheral nerve function were carried out on 29 epileptic patients on long-term anticonvulsant therapy. All but three patients showed abnormalities in one or more electrophysiological measurements, the main abnormality being in amplitude of sensory nerve action potential--this was reduced or absent in 76% of patients. All patients had low concentrations of folate in serum and CSF, these being below the normal ranges in 19 patients. These 19 patients were treated with folate, either 5-formyltetrahydrofolate (10 patients) or folic acid (9 patients), over a period of one month. After therapy all patients had normal levels of folate in serum and CSF, slightly higher levels in CSF being obtained in those receiving 5-formyltetrahydrofolate. Folate therapy significantly reversed abnormalities in motor and sensory nerve distal latencies; the effect was greater with 5-formyltetrahydrofolate, apparently because this produced higher CSF folate concentrations than folic acid. We conclude that folate deficiency may be involved in the development of peripheral neuropathy due to anticonvulsants.
Adult↗