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PubMed1979

Structural role of the polyglutamate portion of the folate found in T4D bacteriophage baseplate.

Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.

Carboxypeptidases↗
PubMed2005

Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study.

OBJECTIVE: To investigate the contribution of red blood cell (RBC) methotrexate polyglutamates (MTX PGs), RBC folate polyglutamates (folate PGs), and a pharmacogenetic index to the clinical status of patients with rheumatoid arthritis treated with MTX. METHODS: 226 adult patients treated with weekly MTX for more than 3 months were enrolled at three sites in a multicentred cross sectional observational study. Clinical status was assessed by the number of joint counts, physician's global assessment of disease activity, and a modified Health Assessment Questionnaire (mHAQ). RBC MTX PG and folate PG metabolite levels were measured by high performance liquid chromatography fluorometry and radioassay, respectively. A composite pharmacogenetic index comprising low penetrance genetic polymorphisms in reduced folate carrier (RFC-1 G80A), AICAR transformylase (ATIC C347G), and thymidylate synthase (TSER*2/*3) was calculated. Statistical analyses were by multivariate linear regression with clinical measures as dependent variables and metabolite levels and the pharmacogenetic index as independent variables after adjustment for other covariates. RESULTS: Multivariate analysis showed that lower RBC MTX PG levels (median 40 nmol/l) and a lower pharmacogenetic index (median 2) were associated with a higher number of joint counts, higher disease activity, and higher mHAQ (p<0.09). Multivariate analysis also established that higher RBC folate PG levels (median 1062 nmol/l) were associated with a higher number of tender and swollen joints after adjustment for RBC MTX PG levels and the pharmacogenetic index (p<0.05). CONCLUSION: Pharmacogenetic and metabolite measurements may be useful in optimising MTX treatment. Prospective studies are warranted to investigate the predictive value of these markers for MTX efficacy.

Aged↗
PubMed1981

The role of methotrexate and dihydrofolate polyglutamates in the enhancement of fluorouracil action by methotrexate.

The effect of the methotrexate (MTX)-5-fluorouracil (FUra) combination on L1210 cell viability was highly sequence dependent in that a 1.4 log (25-fold) increase in cytotoxicity was observed when the MTX preceded FUra, as compared to the reverse sequence. The formation of ternary complexes of thymidylate synthetase has been examined as a basis for the interaction of FUra and MTX in L1210 cells. L1210 cells converted 39% of the total intracellular MTX into MTX-poly-gamma-glutamates within 4 hours of a 1 microM MTX exposure. MTX-diglutamate (2,4-diamino,N 10-methylpteroyl-diglutamate) and MTX-triglutamate were the predominant metabolites. In contrast to the ternary complexes formed with MTX and MTX-diglutamate, the 5-fluorodeoxy-uridylate (FdUMP)-7,8-dihydropteroylpentaglutamate-enzyme and the FdUMP-5,10-methylenetetrahydropteroylpentaglutamate-enzyme complexes were stable to polyacrylamide gel electrophoresis under nondenaturing conditions. MTX-diglutamate enhanced the extent of tight-binding inhibition of thymidylate synthetase activity by FdUMP in the presence of saturating 5,10-methylenetetrahydropteroylpentaglutamate, suggesting that MTX-diglutamate did not antagonize the formation of FdUMP-5,10-methylenetetrahydropteroylpentaglutamate enzyme complex. We propose that the sequence-dependent effect of MTX plus FUra on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase, and consequently a trapping of intracellular folates as dihydrofolate polyglutamates that could increase the extent of FdUMP binding to thymidylate synthetase.

Animals↗
PubMed1995

Quinazoline thymidylate synthase inhibitors: methods for assessing the contribution of polyglutamation to their in vitro activity.

Many quinazoline thymidylate synthase (TS) inhibitors undergo intracellular metabolism to polyglutamate forms which can significantly alter their activity and pharmacodynamics through improved TS inhibition and drug retention. When a series of quinazolines was tested for inhibitory activity towards TS (IC50 0.001-2 microM) and the growth of L1210 cells (IC50 0.005-10 microM), no direct correlation was observed. However, a very good correlation was apparent if a L1210 variant cell line (L1210: RD1694) was used. This line is deficient in its ability to form antifolate polyglutamates. A number of other intact cell methods have also been developed which estimate the contribution that intracellular polyglutamation makes to a compound's activity. These assays were validated using a series of quinazoline-based TS inhibitors with well-defined activity for TS, folypolyglutamate synthetase (FPGS) and the reduced-folate cell membrane carrier (RFC). Short-exposure growth-inhibition assays or the measurement of TS activity in situ after various incubation times, followed by different lengths of time in drug-free medium, can indicate both the speed and extent of appearance of retentive forms (usually polyglutamates). Continuous-exposure growth-inhibition assays, in the presence of leucovorin (LV), are also useful, since only the growth-inhibitory potency of polyglutamated analogues is significantly decreased by LV. Highly polyglutamated compounds, e.g. ZD1694, are virtually inactive in the presence of a high concentration of LV. It is proposed that these methods, when considered together, provide a greater degree of information concerning the rate and extent of polyglutamation of a particular compound than isolated FPGS assays alone.

Animals↗
PubMed1981

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.

Cell Transformation, Viral↗
PubMed1988

Affinity chromatography of naturally occurring folate derivatives.

In a previous report (1980, in Methods in Enzymology, Vol. 66, p. 686, Academic Press, New York) we described the preparation and use of purified milk folate-binding protein covalently linked to a Sepharose matrix. The present study was undertaken to test the capacity of this preparation to purify quantitatively folates from tissue extracts. In experiments that employed tissue extracts containing biologically synthesized [3H]folates, recovery of radioactivity from columns of the immobilized folate-binding protein (affinity columns) was consistently 90-95%. Folates eluted from the affinity column were practically pure. Anion-exchange chromatography of the purified fraction from rat liver extract yielded a number of uv-absorbing peaks which corresponded to the elution profile based on 3H counts, with respect to both the position and the area of the peaks. Treatment of the original extracts with folylpolyglutamate hydrolases resulted in the shift of these peaks to lower retention times, which corresponded to mono- or diglutamyl folate derivatives. Similar results were obtained with extracts from Lactobacillus casei. The purification of tissue folates by affinity chromatography allows determination of folate activity by direct physiochemical methods, which is particularly useful for analysis of folate composition in tissues.

Animals↗
PubMed1980

Folate assay: a comparison of radioassay and microbiological methods.

Folate mono- and polyglutamate standards were assayed by a microbiological method and by four commercially available radioassay methods to evaluate the usefulness of radioassay techniques for the quantitation of naturally occurring folates. Folate monoglutamates exhibited different responses in the radioassay procedures, depending on the one-carbon constituent and oxidation state. Folate polyglutamates exhibited an increased response that varied depending on the folate concentration. The varied responses of folate mono- and polyglutamates in the radioassay procedures make this technique unsuitable for the determination of the mixture of folate derivatives that are normally encountered in biological extracts.

Biological Assay↗
PubMed1980

Electrophoretic identification of poly-gamma-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes.

A method has been developed to characterize the poly-gamma-glutamates of 5,10-methylenetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy[3H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-gamma-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates.

Chemical Phenomena↗
PubMed1980

Utilization of [2-14C]tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid as substrates for folate polyglutamate synthesis in fruit bats: effect of vitamin B-12-deficiency.

[2-14C]Tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid were given intraperitoneally to fruit bats. Folate polyglutamates were formed in the liver from both substrates in different amounts and at different rates. The methylfolate pool appeared to remain separate from the tetrahydrofolate pool. More polyglutamate was formed from tetrahydropteroylglutamic acid than from 5-methyltetrahydropteroylglutamic acid. There was a fall in the folate content of the liver in the vitamin B-12-deficient bat and a more rapid incorporation of folates into polyglutamates but thereafter a more rapid loss of the labelled folate from liver.

Animals↗
PubMed1989

Quinazoline antifolates inhibiting thymidylate synthase: synthesis of four oligo(L-gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid and their enzyme inhibition.

The synthesis is described of four oligo(gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid containing a total of two, three, four, and five L-glutamic acid residues. The tert-butyl group was chosen as the carboxyl protecting group in order to obviate the use of alkali and thus the possibility of gamma----alpha transpeptidation. The starting material, di-tert-butyl glutamate, was coupled to N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester via a mixed anhydride with isobutyl chloroformate. Hydrogenolysis of the benzyloxycarbonyl group in the product gave a carboxyl-protected diglutamate, which either was acylated with 4-[(benzyloxycarbonyl)amino] benzoyl chloride to give a protected aminobenzamide or was cycled further by using the above mixed anhydride/hydrogenolysis sequence into tri-, tetra-, and pentaglutamates. Each of the last named was also acylated, as above, to give a benzamide. The benzyloxycarbonyl group in the benzamides was removed by hydrogenolysis and the amino groups thus exposed were N-alkylated with propargyl bromide. The resulting proparglyamines were further alkylated with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide to give the antifolate poly(t-Bu) esters. Deprotection with trifluoroacetic acid in the final step delivered the desired antifolates as their trifluoroacetate salts. The di- to pentaglutamates were, respectively, 31-, 97-, 171-, and 167-fold more inhibitory to WI-L2 human thymidylate synthase than the parent compound.

Cell Line↗
PubMed1980

The identification of the folate conjugates found in rat liver 48 h after the administration of radioactively labelled folate tracers.

About 70% of the radioactivity retained in the livers of rats dosed 48 h earlier with radioactively labelled folate was incorporated into two folate conjugates. The major derivative was purified and isolated by Sephadex G-15, DEAE-cellulose and DEAE-Sephadex ion-exchange column chromatography and paper chromatography. It was identified as 10-formylpteroylpentaglutamate by a combination of spectral, microbiological, chemical and chromatographic techniques. The minor conjugate, though less well characterized, exhibited similar properties and was assigned the structure 10-formylpteroyltetraglutamate. 10-Formylpteroylpentaglutamate (2.0nmol/g) and 10-formylpteroyltetraglutamate (0.25nmol/g) comprised about 20% of the total endogenous hepatic folate as determined by microbiological assay (Lactobacillus casei after conjugase treatment.

Animals↗