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PubMed1998

58K, a microtubule-binding Golgi protein, is a formiminotransferase cyclodeaminase.

58K was previously identified as a rat liver protein that binds microtubules in vitro and is associated with the cytoplasmic surface of the Golgi apparatus in vivo (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083-16092). We now report that 58K is a formiminotransferase cyclodeaminase (FTCD), a bifunctional enzyme that catalyzes two consecutive steps in the modification of tetrahydrofolate to 5,10-methenyl tetrahydrofolate. Comparative immunoblotting using several monoclonal antibodies made against 58K and a polyclonal antibody made against a chicken liver protein (p60) with similar properties (Hennig, D., Scales, S. J., Moreau, A., Murley, L. L., De Mey, J., and Kreis, T. E. (1998) J. Biol. Chem. 273, 19602-19611) demonstrated precise co-purification of protein recognized by all antibodies through multiple fractionation steps, including gel filtration and ion exchange chromatography, and sucrose gradient ultracentrifugation. Eight peptides derived from 58K showed high sequence identity to amino acid sequences predicted by full length cDNA for p60 and porcine liver FTCD. Furthermore, purified 58K was associated with formiminotransferase and cyclodeaminase activities. Based on these collective results, 58K was concluded to be a rat liver version of FTCD. Microtubules assembled from brain tubulin, but not from liver tubulin, were able to bind rat liver FTCD. Binding to brain microtubules is suspected to occur via polyglutamates that are added post-translationally to tubulin in brain, which was shown to contain very low levels of FTCD, but not to tubulin in liver, which was determined to be the richest tissue source, by far, of FTCD. The physiological significance of the microtubule binding activity of FTCD is thus called into question, but an association of FTCD with the Golgi apparatus has now been established.

Amino Acid Sequence↗
PubMed1983

A structural role for dihydropteroyl hexaglutamate in the tail baseplate of various bacteriophages.

A novel non-metabolic role is proposed for dihydropteroyl hexaglutamate as a critical link binding together sub-structures of the tail of Escherichia coli bacteriophage T4. Six molecules of this folate compound have been found to be components of the complex tail baseplate of the phage particle. The baseplate is assembled using a total of at least 18 viral gene products in a series of reactions in which six wedge-like elements (each 0.7 X 10(6) daltons) bind symmetrically around a central tail plug (1.55 X 10(6) daltons) to form a flat hexagonal structure. It appears likely that the pteridine portion of the folate binds to a site on a viral-induced dihydrofolate reductase molecule, a wedge component, while the glutamate residues of the folate bind to a viral-induced thymidylate synthase molecule, a central plug component. Additionally, it appears that the folyl glutamate residues play a role in forming a flexible bond between the proximal end of the phage long tail fiber and the baseplate. Two bacteriophages attacking a quite different bacterial host, Pseudomonas syringae, have been isolated and partially characterized. Both phage strains have tail structures morphologically analogous to T4. Both were irreversibly inactivated by an enzyme which cleaves the gamma-glutamyl bonds of folyl polyglutamate. It appears that these Pseudomonas phage particles also contain a folyl poly-glutamate whose integrity is essential for their infectivity.

Escherichia coli↗
PubMed1995

Osteopontin and related phosphorylated sialoproteins: effects on mineralization.

Osteopontin is one of a family of phosphorylated sialoproteins found in the extracellular matrices of mineralized connective tissues. Solution studies from a variety of laboratories have shown that while some of these proteins (e.g., osteopontin, dentin sialoprotein) inhibit calcium phosphate production, others, such as bone sialoprotein, can act as a nucleator of calcium phosphate formation. The differences in the behaviors of these proteins can be related to their interactions with mineral crystal nuclei and crystal growth sites. The specificity of these interactions, in turn, can be related to differences in the primary structures of the sialoproteins and to the extent to which they are phosphorylated. In vitro systems for the study of osteopontins and osteopontin-related protein effects on mineral deposition are described, along with mechanisms explaining the contrasting abilities of the polyglutamate-containing bone sialoprotein to act as a nucleator, while the polyaspartate-containing osteopontin and dentin sialoproteins inhibit calcium phosphate formation and growth.

Amino Acid Sequence↗
PubMed1982

The effect of derivatives of folic acid on the fluorodeoxyuridylate-thymidylate synthetase covalent complex in human colon xenografts.

This study was designed to examine the endogenous concentrations of 5,10-methylenetetrahydrofolate (CH2FH4) in human colorectal adenocarcinoma xenografts, and to determine the ability of other folate derivatives to increase the formation of the ternary covalent complex between CH2FH4, [6-3H]-5-fluorodeoxyuridylate (FdUMP) and thymidylate synthetase (TS, EC 2.1.1.45). Levels of CH2FH4 were determined by measuring the release of [3H]2O from [5-3H]-dUMP using TS from Lactobacillus casei. The reaction was linear from 1.9 X 10(-13) to 2.4 X 10(-11) mol of CH2FH4 assayed. Concentrations of CH2FH4 were low, ranging from 66 to 233 nM in cell water. Tetrahydrofolate (FH4) and dihydrofolate (FH2) increased complex formation, while 5-formyltetrahydrofolate (5-CHOFH4) and 5-methyltetrahydrofolate (5-CH3FH4) decreased the covalent binding of [6-3H]-FdUMP in vitro. Administration of FH4 or FH2 to tumor-bearing mice reduced subsequent formation of the covalent complex in vitro. Since 5-CH3FH4 is a major derivative of folate in mammalian tissues, its effect on the covalent binding of [6-3H]-FdUMP was examined further; even in the presence of homocysteine and cyanocobalamin (B12), the formation of the covalent complex was not increased. The fate of [5-14CH3]-FH4 was subsequently examined in vivo. In tumors at 1 hr after injection, 72% of the radiolabel remained as [5-14CH3]-FH4, while 17% had been converted to [14C]-methionine or incorporated into protein. By contrast, however, the incorporation of radiolabel into the protein fraction of liver was almost 30-fold greater at this time. At 4 hr, radioactivity in tumors (dpm/g) and in the fraction associated with [5-14CH3]-FH4 was decreased by over 60%, while metabolism was increased by only 13%. No polyglutamate forms of [5-14CH3]-FH4 were detected in tumors at 4 hr after treatment.

Adenocarcinoma↗
PubMed1998

Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

Animals↗
PubMed1994

Novel pyrrolo[2,3-d]pyrimidine antifolate TNP-351: cytotoxic effect on methotrexate-resistant CCRF-CEM cells and inhibition of transformylases of de novo purine biosynthesis.

N-[4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidine ring, is a novel antifolate that exhibits potent antitumor activities against mammalian solid tumors. The mechanism of action of TNP-351 was evaluated using some methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as partially purified enzymes folylpolyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleotide transformylase (AICARTFase), and glycinamide ribonucleotide transformylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to inhibit the growth of L1210 and CCRF-CEM cells in culture, with the doses effective against 50% of the cells (ED50 values) being 0.79 and 2.7 nM, respectively. The growth inhibition caused by TNF-351 was reversed by leucovorin or a combination of hypoxanthine and thymidine. The methotrexate-resistant CCRF-CEM cell line, which has an impaired methotrexate transport, showed less resistance to TNP-351 than to methotrexate. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (Km) of 1.45 microM and a maximum of velocity (Vmax) of 1,925 pmol h-1 mg-1. Inhibitory activities of TNF-351-Gn (n = 1-6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (Ki): G1, 52 microM; G6, 0.07 microM]. Neither TNP-351 nor its polyglutamates were very strong inhibitors of GARTFase. These findings have significant implications regarding the mechanism of action of TNP-351.

Acyltransferases↗
PubMed2000

Polyglutamation of antifolates is not required for induction of extracellular release of adenosine or expression of their anti-inflammatory effects.

Methotrexate (MTX) exerts an anti-inflammatory effect, reportedly by enhancing the release of adenosine, through an accumulation of 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR). To examine the role of polyglutamation in promoting anti-inflammatory effects by antifolates, we tested whether a new antifolate designed to be resistant to intracellular polyglutamation (MX-68) exhibited anti-inflammatory effects and stimulated adenosine release. Both MX-68 and MTX (at concentrations greater than 0.1 microM) increased the release of adenosine from Daudi cells in vitro. Inhibition of AICAR synthesis suppressed adenosine release by MX-68 and MTX. The anti-inflammatory effects of antifolates were estimated using the murine air pouch model, in which a BALB/c mouse was intraperitoneally injected with MX-68 or MTX once a week for 3 weeks. MX-68 (0.5 mg kg(-1) week(-1)), as well as MTX, inhibited infiltration of leukocytes into the air pouch. This inhibitory effect was suppressed in the presence of an adenosine A(2) receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). These results suggest that MX-68, like MTX, exerts anti-inflammatory effects through the accumulation of AICAR and release of adenosine. These results suggest that polyglutamation of antifolate is not required for expression of anti-inflammatory effects.

2-Aminoadipic Acid↗
PubMed1983

New methods for the study of folate coenzymes: endogenous polyglutamate patterns of subcellular hepatocyte fractions and of regenerating rat liver.

A procedure has been developed that permits the quantitation of three different pools of one-carbon-substituted folates with the simultaneous determination of their corresponding poly-gamma-glutamyl chain lengths. Pool 1 is made up of N5,N10-methylene-tetrahydrofolates and unsubstituted tetrahydro and dihydrofolates. Pool 2 is made up solely of N5-methyl-tetrahydrofolates and pool 3 includes the folates with one-carbon-substituents at the formyl oxidation level: N5,N10-methenyl-tetrahydrofolates, N10- and N5-formyl-tetrahydrofolates, and N5-formimino-tetrahydrofolates. Conditions have been found that permit the selective cleavage of the C9-N10 bond of the folates of pools 1, 1 + 2, and 1 + 2 + 3. The cleaved folates are quantitated as the Bratton-Marshall azo-dyes of the p-amino-benzoyl-poly-gamma-glutamates (AzoGlun) after converting the uncleaved pools to Bratton-Marshall negative products. Determination of the poly-gamma-glutamyl chain length of the cleaved pools is carried out by high pressure liquid chromatography of the AzoGlun comparing the elution position of each peak with that of authentic synthetic markers. Quantitation of the individual peaks is obtained by integration and by reference to an internal synthetic standard (usually AzoGlu3). To increase the sensitivity of the procedure and make it applicable to plasma or needle biopsy specimens we have developed a synthesis for [3H]-N'-naphthylethylenediamine. This compound is the one coupled to the diazotized p-aminobenzoyl-poly-gamma-glutamates to form the AzoGlun in the Bratton-Marshall test. The new methods do not require labeling the animal's folates by the prior injection of radioactive folic acid thus permitting the study of endogenous folate patterns. Applied to rat liver these methods have shown that pool 3 accounts for 44.3 +/- 7.7%, pool 2 for 36.7 +/- 4.7%, and pool 1 for 18.5 +/- 5.8% of the total folates. The pentaglutamates made up about 52% and the hexaglutamates about 42% of the folates. Hepta and tetraglutamates account for approximately 2.8 and 3.5% respectively. The cytosolic folates are predominantly hexaglutamates whereas pentaglutamates predominate in the heavy mitochondrial fraction. Profound changes in the relative proportions of the polyglutamates were observed 24 h after partial hepatectomy. There are marked increases in hepta and hexaglutamates and decreases in penta and tetraglutamates. These results support the hypothesis that changes in polyglutamate chain length are related to the regulation of one-carbon metabolism.

Animals↗
PubMed2004

Gateways to clinical trials.

Gateways to Clinical Trials is a guide to the most recent clinical trials in current literature and congresses. The data in the following tables has been retrieved from the Clinical Studies Knowledge Area of Prous Science Integrity(R), the drug discovery and development portal, http://integrity.prous.com. This issue focuses on the following selection of drugs: Activated protein C concentrate, Ad-CD154, Adeno-Interferon gamma, alemtuzumab, APC-8024, 9-aminocamptothecin, aprepitant, l-arginine hydrochloride, aripiprazole, arsenic trioxide, asimadoline; O6-Benzylguanine, bevacizumab, Bi-20, binodenoson, biphasic insulin aspart, bivatuzumab, 186Re-bivatuzumab, BMS-181176, bosentan, botulinum toxin type B, BQ-123, bryostatin 1; Carboxy- amidotriazole, caspofungin acetate, CB-1954, CC-4047, CDP-860, cerivastatin sodium, clevidipine, CTL-102; 3,4-DAP, darbepoetin alfa, decitabine, desloratadine, DHA-paclitaxel, duloxetine hydrochloride; Efalizumab, EGF vaccine, eletriptan, eniluracil, ENMD-0997, eplerenone, eplivanserin, erlosamide, ertapenem sodium, escitalopram oxalate, esomeprazole magnesium, eszopiclone, everolimus, exatecan mesilate, exenatide, ezetimibe; Fondaparinux sodium, FR-901228, FTY-720; Gefitinib, gemtuzumab ozogamicin, gepirone hydrochloride; Hexyl insulin M2, human insulin; Imatinib mesylate, insulin detemir, insulin glargine, iodine (I131) tositumomab, ISV-205, ivabradine hydrochloride, ixabepilone; Levetiracetam, levocetirizine, linezolid, liposomal NDDP, lonafarnib, lopinavir, LY-156735; Mafosfamide cyclohexylamine salt, magnesium sulfate, maxacalcitol, meclinertant, melagatran, melatonin, MENT, mepolizumab, micafungin sodium, midostaurin, motexafin gadolinium; Nesiritide, NS-1209, NSC-601316, NSC-683864; Osanetant; Palonosetron hydrochloride, parecoxib sodium, pegaptanib sodium, peginterferon alfa-2a, peginterferon alfa-2b, pegylated OB protein, pemetrexed disodium, perillyl alcohol, picoplatin, pimecrolimus, pixantrone maleate, plevitrexed, polyglutamate paclitaxel, posurdex, pramlintide acetate, prasterone, pregabalin; Rasburicase, rimonabant hydrochloride, rostaporfin, rosuvastatin calcium; SDZ-SID-791, sibrotuzumab, sorafenib, SU-11248; Tadalafil, targinine, tegaserod maleate, telithromycin, TheraCIM, tigecycline, tiotropium bromide, tipifarnib, tirapazamine, treprostinil sodium; Valdecoxib, Valganciclovir hydrochloride, Vardenafil hydrochloride hydrate; Ximelagatran; Zofenopril calcium, Zoledronic acid monohydrate.

Clinical Trials as Topic↗
PubMed1988

[The role of thymidylate synthetase in sequential dose of MTX and 5-FU in the advanced scirrhous type gastric cancer].

The biochemical rationale for the potentiation of the effects of 5-FU by MTX is based on an increased PRPP level or MTX polyglutamate produced by MTX. The cytotoxic action of MTX results not only from inhibition of DHFR but also depends upon thymidylate synthetase (TS), a key enzyme in DNA synthesis. We obtained a monoclonal antibody to TS using a hydrophilic peptide consisting of 20 amino acids in the TS amino acid sequence and demonstrated by PAP that TS was detectable in poorly differentiated adenocarcinoma cells but not in well differentiated adenocarcinoma cells. Upon clinical application of sequential doses of MTX and 5-FU, the median survival durations were 318 days and 156 days for scirrhous-type gastric cancer patients and non-scirrhous-type gastric cancer patients respectively. These results suggest that immunohistochemistry with TS antibody is available as an indicator of the effect of this drug regimen.

Adenocarcinoma, Scirrhous↗
PubMed1983

A pteroylpolyglutamate binds to tetramers in deoxyhemoglobin but to dimers in oxyhemoglobin.

The binding of a physiological concentration of pteroylhepta(glutamate) to oxy- and deoxyhemoglobin in large excess was measured by ultrafiltration. The variation of free to bound folate with hemoglobin concentration showed that a single molecule of the pteroylpolyglutamate is bound by deoxyhemoglobin tetramers and by alpha beta dimers in oxyhemoglobin. Although the binding sites are different, the affinity constants are the same and very similar to the 2,3-bisphosphoglycerate binding energy. Nevertheless, in view of the small proportion of dimers in oxyhemoglobin much more pteroylhepta(glutamate) is bound by deoxyhemoglobin over a wide range of hemoglobin concentrations. Because even 2% deoxyhemoglobin is enough to bind all of the erythrocyte folate as polyglutamate, the bulk of it will be bound at physiological oxygen pressures. Free folate could only be expected in fully oxygenated erythrocytes. Therefore, the reaction of pteroylpolyglutamates with hemoglobin represents an oxygenation-dependent storage mechanism that can account for the 40-fold excess of the vitamin in the erythrocyte over the amounts in the serum. Because methotrexate is also converted to polyglutamate derivatives in the erythrocyte, this drug is likely to be concentrated and stored there by the same mechanism.

Folic Acid↗
PubMed1991

Effects of choline deficiency and methotrexate treatment upon liver folate content and distribution.

We examined the effects of feeding rats a choline deficient diet, of treating rats with low doses of methotrexate (MTX, 0.1 mg/kg, daily), and of combined choline deficiency and MTX treatment upon the content and distribution of folates in liver. We used a newly devised technique for analysis of folates which utilized affinity chromatography followed by high pressure liquid chromatography. Compared to control rats, total hepatic folate content decreased by 31% in the choline deficient rats, by 48% in the MTX treated rats, and by 60% in rats which were both choline deficient and treated with MTX. In extracts of livers from control rats, folates were present predominantly as penta (35%) and hexaglutamyl (52%) derivatives. The pteridine ring structure distribution of these folates was as follows: 48% 5-methyltetrahydrofolate, 14% formylated tetrahydrofolate, and 39% tetrahydrofolate. In choline deficient animals, there was a decrease in the relative concentration of pentaglutamyl folates and an increase in the relative concentration of heptaglutamyl folates. In livers from MTX treated animals, MTX-polyglutamates with 2-5 glutamate residues accumulated. The consequences of MTX treatment were: a) an elongation of the glutamate chains of the folates as the proportion of hepta- and octaglutamyl derivatives was increased relative to penta- and hexaglutamyl folates; b) the occurrence of unreduced folic acid; c) a decrease in the relative concentration of 5-methyltetrahydrofolate and an increase in the relative concentration of formylated tetrahydrofolate, and d) no change in the relative concentrations of tetrahydrofolate. In livers from animals that were both choline deficient and treated with MTX, the tetrahydrofolate concentrations were 50% of control while formylated tetrahydrofolate concentrations increased 3-fold. These data are discussed from the standpoint of the current understanding of mechanisms that regulate the elongation of the glutamic acid chains of folates and those that regulate folate dependent synthesis and utilization of one carbon unit.

Animals↗
PubMed1983

A quinazoline antifolate as inhibitor of folate metabolism in mouse L-cells.

The effects of quinazoline antifolate CB 3703 on folate metabolism in mouse L-cells were examined by measuring the amount of 14C label in folate derivatives and its distribution in separated folylmono- and polyglutamates in the cells grown in the presence of radioactive folate and quinazoline. The total radioactivity of folyl derivatives in the quinazoline-treated cells did not exceed 15% of the radioactivity extracted from the control cells, in spite of the fact that quinazoline CB 3703 did not interfere with the uptake of folate into L-cells. Analysis of folylmono- and polyglutamate derivatives, either incubated or not incubated with gamma-glutamylcarboxypeptidase, by chromatography on Sephadex G-25, G-15 or DEAE-cellulose revealed that in the quinazoline-treated cells practically all radioactivity was found in the position of unmetabolized folate. It seems probable, therefore, that quinazoline CB 3703 impairs in L-cells folate reduction and conversion to its coenzymatic forms and their subsequent polyglutamation. These facts seem to be responsible for a decrease in retention of folate derivatives in the quinazoline CB 3703-treated cells.

Animals↗
PubMed1993

Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism.

The effect of folylpoly-gamma-glutamate synthetase (FPGS) levels on folate accumulation was investigated in Chinese hamster ovary cells expressing various levels of human and Escherichia coli FPGS activity. At low medium folate concentrations, folate accumulation was limited by influx and was independent of FPGS activity except in cells expressing extremely low levels of FPGS. Essentially all transported folate was metabolized to retained polyglutamate derivatives, the chain length of which varied with the level of FPGS activity. As medium folate concentration increased through the physiological to the pharmacological range, cellular folate accumulation became proportional to FPGS activity and the chain length of intracellular folates decreased. At high folate concentrations, competition between substrates for FPGS limited the extent of polyglutamylation and less than 5% of transported folate was retained by the cell. Pteroyltriglutamates functioned as effectively as the longer chain length polyglutamates normally found in mammalian cells in the metabolic cycles of de novo purine and thymidylate biosynthesis but were unable to support glycine and methionine synthesis. Transfectants expressing human FPGS and containing folates of glutamate chain length ranging from four to eight were equally effective at supporting glycine synthesis, and transfectants expressing higher levels of FPGS were able to grow in the absence of methionine. Growth in the absence of methionine required high (nonphysiological) intracellular folate levels and longer chain length polyglutamates.

Animals↗
PubMed1971

Studies on the absorption and metabolism of folic acid. I. Folate absorption in the dog after exposure of isolated intestinal segments to synthetic pteroylpolyglutamates of various chain lengths.

Folic acid absorption was studied in anesthetized dogs by determining the amount and chemical nature of folate in venous blood emerging from isolated intestinal segments containing free folic acid and/or pteroylpolyglutamates of a known chain length. Chromatographically pure test materials placed in the lumen were prepared by unambiguous solid phase synthetic methods. This synthetic procedure not only yields compounds of known structure, it also provides a means by which glutamic acid residues at any given position in the gamma glutamyl chain can be made radioactive. For example, teropterin (pteroyltriglutamate) was synthesized in such a way that (14)C was present only in the middle glutamic acid unit. Suitable placement of label permitted assessment of the extent of peptide cleavage. The action of plasma conjugase was inhibited by copper chloride. Plasma samples were analyzed by Lactobacillus casei and Streptococcus faecalis assay, by column chromatography, and by quantitative measurement of pteridine-bound radioactivity. It was observed that biologically active folate appeared in the mesenteric vein with either pteroylmono-, di-, tri-, penta-, or heptaglutamate in the lumen. Generally speaking the absorption rate appeared to be inversely related to the length of the gamma glutamyl side chain. Roughly twice as much folic acid appeared in the circulation from (3)H-labeled pteroylmonoglutamate as from (14)C-labeled pteroylpentaglutamate when equimolar amounts of each were placed simultaneously in a single intestinal segment. Pteroylmonoglutamate appeared to be the predominant form entering the blood from each of the precursors tested. However, evidence was obtained that pteroyldiglutamate may enter the mesenteric vein soon after placing pteroyldi-, or triglutamate in the lumen, but not with the higher polyglutamates. Comparison of radioactivity and biological activity patterns suggests little conversion, if any, to reduced or methylated forms during the first 30 min of passage through the intestinal mucosa. We conclude that both pteroylmonoglutamates and pteroyldiglutamates may across the intestinal mucosa of the dog, and that reduction and methylation are not essential to the absorption process.

Animals↗
PubMed1989

The enhancement of the activity of 10-propargyl-5,8-dideazafolate and 5,10-dideazatetrahydrofolate by inhibitors of dihydrofolate reductase.

Treatment of H35 hepatoma cells with the lipid soluble dihydrofolate reductase inhibitors metoprine and trimetrexate cause a nearly 10-fold increase in the toxicity of the antipyrimidine folate analogue PDDF and the antipurine folate analogue DDATHF. Evaluation of these interactions by the combination index developed by Chou (17-20) yields results conforming to synergistic interactions. The capacity of PDDF to inhibit thymidylate synthase in intact cells as measured by tritium release from [5-3H]deoxyuridine was increased by approximately the same amount by preincubation with the dihydrofolate reductase inhibitors. The primary effect of the reductase inhibitors in causing greater activity may be a reduction in cellular folates which can cause 5,10-CH2H4PteGlun to decrease and cellular PDDF (polyglutamates) to increase. These conditions would favor inhibition of thymidylate synthase by PDDF by promoting formation of the stable, inhibited PDDF (polyglutamates)-thymidylate synthase-dUMP complex (12).

Animals↗
PubMed1991

5-Formyltetrahydrofolate polyglutamates are slow tight binding inhibitors of serine hydroxymethyltransferase.

The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods. These methods included: determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics. Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex. However, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate. The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase. The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one-carbon metabolism.

Animals↗