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Query: UNIPROT:P06889 (Mol)
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1. Synthesis of polyglutamate derivatives from radioactively labelled folic acid, folinic acid and methotrexate has been studied in human phytohaemagglutinin-transformed lymphocytes. 2. With labelled folic acid as the precursor, approximately 20% of the labelled cell folate was found in each of the mono-, tetra-, penta- and hexaglutamate peaks after 72 h of incubation. At earlier times, the amounts of labelled folate polyglutamates were proportionately greater and the amount of labelled higher folate polyglutamates was lower. After only 4 h incubation over 80% of the intracellular labelled folate was still in the monoglutamate form. 3. With labelled folinic acid used as precursor, approximately 30% of labelled cell folate after 72 h incubation was in the form of folate pentaglutamate, with tri- and tetra-glutamate forming the other major peaks. The speed of formation of polyglutamate derivatives was substantially more rapid than from folic acid. 4. Methotrexate was found to decrease considerably the amount of folate polyglutamate formation from labelled folic acid in lymphocytes byt nevertheless some di-, tri- and traces of tetra-glutamate were formed. On the other hand, methotrexate had no effect on folate polyglutamate formation with labelled folinic acid used as the precusor. 5. Polyglutamate derivatives of labelled methotrexate were formed by human lymphocytes with mean amounts of 4-5% of di-, 1-5% of tri-, 1-0% of tetra- and 0-7% of penta-glutamate derivatives being formed over the period 48-72h of culture. 6. Pentaglutamate derivatives probably constitute the largest group of intracellular folates in human cells but a complex mixture exists. 7. The enzyme synthesizing polyglutamate derivatives of folate in human cells prefers a reduced folate as substrate but the requirement for a reduced form is not absolute. The nature of the reduced folate is uncertain but it is suggested on the basis of previous work that tetrahydrofolate rather than methylhydropfolate or any other reduced folate monoglutamate is the preferred substrate.
Clin Sci Mol Med 1976 Jan
PMID:Synthesis of folate polyglutamates in human cells. 108 4

The relationship between vitamin B12 and folate and the effect of methionine on folate metabolism during B12 deficiency in rats is best explained by the prevention of the accumulation of 5-methyl-H4PteGlu by vitamin B12 and/or methionine. Although several points remain to be clarified, the 'methyl trap' hypothesis provides the most satisfactory explanation for the relation between vitamin B12, methionine and folic acid. This concept is extended by the hypothesis that H4PteGlu is the most active substrate for pteroylpolyglutamate synthetase, and thus accounts for the effect of methionine or vitamin B12 increasing liver folate levels.
Mol Cell Biochem 1975 Nov 14
PMID:The relationships between vitamin B12 and folic acid and the effect of methionine on folate metabolism. 119 3

The effect of the inhibition of dihydrofolate reductase by methotrexate on the cellular folates involved in de novo purine and thymidylate biosynthesis has been measured in H35 hepatoma cells grown in 4 microM folic acid or 20 nM folinic acid. The major cellular folate species in cells from medium with folate or folinate is 10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of 5,10-methylenetetrahydrofolate and tetrahydrofolate. Cultures were exposed to a pulse dose of methotrexate, resulting in the accumulation of nearly exclusively methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced folate coenzymes, which generally corresponded to the extent of inhibition of glycine and deoxyuridine incorporation. This was accompanied by an increase of the cellular dihydrofolate and 10-formyldihydrofolate. In the H35 cells the effect of methotrexate polyglutamates on the reduced folate coenzyme pools was restricted to dividing cultures, because the reduced folate coenzymes were not depleted in confluent cultures. The results demonstrate that the methotrexate and methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit dihydrofolate reductase but are not adequate to inhibit thymidylate synthase and prevent the depletion of reduced folate coenzymes. Thus, inhibition of de novo glycine and deoxyuridine incorporation into DNA as a result of dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate, the respective folate coenzymes for de novo purine and thymidylate synthesis.
Mol Pharmacol 1992 Nov
PMID:Depletion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate by methotrexate in cultured hepatoma cells. 143 54

The measurement of urinary 6 beta-hydroxycortisol (6 beta-OHF) has been widely used as a non-invasive clinical test to detect cytochrome P450 induction. Although only a minor biotransformation, 6 beta-OHF formation represents a sensitive target for many P450-inducing drugs and environmental chemicals in man. There is good evidence that an isozyme of the P450IIIA subfamily is predominantly responsible for 6 beta-hydroxylase activity and therefore it has been suggested that urinary 6 beta-OHF is a marker of the induction of P450IIIA. The basis of the present study was that in order to realistically assign to 6 beta-OHF the status of a P450IIIA marker we should characterize all the metabolites of cortisol produced by human liver and assess inter-liver variability. Incubations at 37 degrees C for 2 h contained [3H]cortisol (0.1 microCi, 1 or 50 microM), MgCl2 (10 mM), microsomal or cytosolic protein (3 mg), an NADPH-regenerating system and 1/15 M phosphate buffer (pH 7.4) to give a final volume of 0.5 ml. Extraction with ethyl acetate (2 x 2 ml) was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the microsomal incubations (n = 6 livers) produced 6 alpha-hydroxycortisol (6 alpha-OHF), 6 beta-OHF, 20 beta-dihydroxycortisol, 20 beta-dihydroxycortisone, cortisone, and 3 alpha, 5 beta-tetrahydrocortisone (3 alpha, 5 beta-THE), while five produced 6 beta-hydroxycortisone and four produced 3 alpha, 5 beta-tetrahydrocortisol (3 alpha, 5 beta-THF). The cytosolic incubations gave a much simpler metabolic profile, with 3 alpha, 5 beta-THF the major metabolite and 3 alpha, 5 beta-THE a minor metabolite. There was considerable inter-individual variability in metabolite profiles from microsomal incubations. 6 beta-OHF varied from 2.8 to 31.7%. Major metabolites were cortisone and 3 alpha, 5 beta-THE. Inter-liver variability was less for cytosolic incubations, the major metabolite always being 3 alpha, 5 beta-THF. In conclusion we have rigorously identified the hepatic metabolites of cortisol formed in vitro. The highly complex and variable hepatic metabolism of cortisol clearly limits the use of urinary 6 beta-OHF excretion as a marker of baseline P450IIIA activity in man.
J Steroid Biochem Mol Biol 1992 Dec
PMID:Cortisol metabolism by human liver in vitro--I. Metabolite identification and inter-individual variability. 147 63

Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine. We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT. The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate. The for+ gene encodes a polypeptide of 479 amino acids which shows significant similarity to amino acid sequences of SHMT from bacterial and mammalian sources (47 and 60% amino acid identity, respectively). The for+ mRNA has several different start and stop sites. The abundance of for+ mRNA increased in response to amino acid imbalance induced by glycine supplementation, suggesting regulation by the N. crassa cross-pathway control system, which is analogous to general amino acid control in Saccharomyces cerevisiae. This was confirmed by documenting that for+ expression increased in response to histidine limitation (induced by 3-amino-1,2,4-triazole) and that this response was dependent on the presence of a functional cross-pathway control-1 (cpc-1) gene, which encodes CPC1, a positively acting transcription factor. There are at least five potential CPC1 binding sites upstream of the for+ transcriptional start, as well as one that exactly matches the consensus CPC1 binding site in the first intron of the for+ gene.
Mol Cell Biol 1992 Apr
PMID:Characterization of the formate (for) locus, which encodes the cytosolic serine hydroxymethyltransferase of Neurospora crassa. 153 27

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.
J Mol Biol 1992 Jul 20
PMID:Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution. 164 Apr 65

5,10-Dideazatetrahydrofolic acid (DDATHF) is a new potent antitumor agent that specifically inhibits purine biosynthesis, primarily through inhibition of glycinamide ribonucleotide transformylase, the first of the tetrahydrofolate-requiring enzymes in the de novo synthesis pathway. DDATHF has been shown to be an excellent substrate for mouse liver folylpolyglutamate synthetase in vitro, suggesting that intracellular conversion to polyglutamates could play an important role in the action of this antifolate. In this report, metabolic studies of the 6R-diastereomer of DDATHF in the cultured human leukemia cell lines CCRF-CEM and HL-60 are presented. At both 1 and 10 microM (6R)-DDATHF was rapidly converted to polyglutamates in both cell lines. DDATHF(Glu)5 and DDATHF(Glu)6 were the main intracellular metabolites. After incubation in drug-free medium, (6R)-DDATHF polyglutamates were better retained intracellularly with increasing glutamate chain length. (6R)-DDATHF showed reduced cytotoxicity toward a folylpolyglutamate synthetase-deficient cell line, CCRF-CEM30/6 related to a dramatically diminished accumulation of polyglutamates. The activity of (6R)-DDATHF in CCRF-CEM30/6 cells was decreased after both short and prolonged exposures. These results suggest that polyglutamylation of (6R)-DDATHF not only represents a mechanism for trapping the drug inside the cells but also produces a more potent inhibitor of the target enzyme.
Mol Pharmacol 1991 Jan
PMID:Intracellular metabolism of 5,10-dideazatetrahydrofolic acid in human leukemia cell lines. 170 76

MTHFD is a folate-dependent trifunctional protein comprised of three activities: N5,N10-methylenetetrahydrofolate dehydrogenase, N5,N10-methenyltetrahydrofolate cyclohydrolase, and N10-formyltetrahydrofolate synthetase. The enzymes catalyze sequential interconversion of tetrahydrofolate derivatives required for purine, methionine, and thymidylate synthesis. A Chinese hamster ovary cell line (Ade-E), reported to have reduced cyclohydrolase activity, was studied to characterize the nature of the mutation. Enzymatic assays showed reduced activities of all three enzymes of the polypeptide. Immunoblotting and immunoprecipitation of radiolabeled cell extracts indicated that MTHFD protein was greatly reduced or absent in the mutant. Northern analysis of a clonal derivative of Ade-E revealed normal levels of MTHFD mRNA. These results suggest that the mutation affects a posttranscriptional process in the synthesis of the trifunctional enzyme.
Somat Cell Mol Genet 1991 Jul
PMID:Deficient synthesis of MTHFD, a trifunctional folate-dependent enzyme, in the CHO Ade E mutant. 188 35

Oral administration of cortisone acetate is widely used to treat prepubertal patients with congenital adrenal hyperplasia (CAH). However, efficient 'first pass' hepatic conversion of the biologically inactive cortisone (E) to cortisol (F) by the 11-reductase component of the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) system is required for suppression of the hypothalamic-pituitary-adrenal (HPA) axis. 11-beta-HSD activity can be assessed by measurement of urinary tetrahydroderivatives of E (tetrahydrocortisone, THE) and F (tetrahydrocortisol, THF), formed in separate hepatic compartments by reduction of the A ring. Inadequate HPA axis suppression is frequently encountered in peripubertal CAH patients receiving cortisone acetate therapy. In this paper, we describe THE and THF concentration in 24 h urine samples collected every 3-6 months from 14 prepubertal patients with simple virilizing CAH. The patients had been receiving cortisone acetate and 9 alpha-fluorohydrocortisone since diagnosis and were investigated for 2-4 years during which there was marked intra- and inter-individual variation in the level of suppression. Good and poor control of HPA axis suppression were defined on the basis of a profile of early morning serum 17-hydroxyprogesterone, androstenedione, plasma renin activity and 24 h urinary excretion of pregnanetriol, pregnanetriolone and 5 beta, 17 alpha-hydroxypregnanolone. Serum steroids were measured by RIA and urinary metabolites quantitated as methyloxime-trimethylsilylimidazole derivatives by gas chromatography and GC-mass spectrometry. There were no significant differences in the THE/THF ratio between male (n = 9) and female (n = 5) patients during either good or poor therapeutic control. The data were therefore analyzed without consideration of patient sex. Urinary THE/THF (mean +/- SD) was significantly higher in patients during periods of poor control (6.56 +/- 2.51, P less than 0.001) compared with periods of good control (3.73 +/- 0.96) in the same patients. THE/THF levels were also significantly (P less than 0.001) higher in CAH patients, irrespective of the level of control, than those for the normal subjects (1.79 +/- 0.20). Furthermore, THE excretion was significantly higher during periods of poor control compared with good control at all doses of cortisone acetate administered (10-50 mg/day). There were no significant differences in THF excretion. THE levels also rose significantly (P less than 0.001) in response to increasing total dose during periods of poor control. The increase in THF excretion was slight and significant only at doses greater than 40 mg/day compared with doses less than 15 mg/day.(ABSTRACT TRUNCATED AT 400 WORDS)
J Steroid Biochem Mol Biol 1991 Oct
PMID:A possible defect in the inter-conversion between cortisone and cortisol in prepubertal patients with congenital adrenal hyperplasia receiving cortisone acetate therapy. 191 35

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.
Mol Microbiol 1991 Jan
PMID:Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum. 201 4


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