Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase

Hillcoat, Brian L.; Nixon, P.G.F.; Blakley, Raymond L.

doi:10.1016/0003-2697(67)90179-0

Cited by 243 publications

(116 citation statements)

References 20 publications

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“…Reactions were run in water-jacketed cuvettes with the temperature maintained at 27°C, and NADPH plus 7,8-DHF consumption monitored at 340 nm with a Cary 300 spectrophotometer. Formation of THF product was calculated using a millimolar extinction coefficient of 12.3 mM Ϫ1 cm Ϫ1 with 7,8-DHF as substrate (55).…”

Section: Methodsmentioning

confidence: 99%

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

Bailey

Ayling

2009

Proc. Natl. Acad. Sci. U.S.A.

335

291

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Numerous clinical trials using folic acid for prevention of cardiovascular disease, stroke, cognitive decline, and neural tube defects have been completed or are underway. Yet, all functions of folate are performed by tetrahydrofolate and its one-carbon derivatives. Folic acid is a synthetic oxidized form not significantly found in fresh natural foods; to be used it must be converted to tetrahydrofolate by dihydrofolate reductase (DHFR). Increasing evidence suggests that this process may be slow in humans. Here we show, using a sensitive assay we developed, that the reduction of folic acid by DHFR per gram of human liver (n ‫؍‬ 6) obtained from organ donors or directly from surgery is, on average, less than 2% of that in rat liver at physiological pH. Moreover, in contrast to rats, there was almost a 5-fold variation of DHFR activity among the human samples. This limited ability to activate the synthetic vitamer raises issues about clinical trials using high levels of folic acid. The extremely low rate of conversion of folic acid suggests that the benefit of its use in high doses will be limited by saturation of DHFR, especially in individuals possessing lower than average activity. These results are also consistent with the reports of unmetabolized folic acid in plasma and urine.folate ͉ provitamin utilization ͉ unmetabolized folic acid ͉ vitamin supplements ͉ nutrition

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Section: Methodsmentioning

confidence: 99%

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

Bailey

Ayling

2009

Proc. Natl. Acad. Sci. U.S.A.

335

291

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“…1800 spectrophotometer. The difference extinction coefficient for the reaction was taken as 12300 litre molh -cm-' (Hillcoat et al, 1967), and the unit of enzyme activity was defined as the amount required to reduce 1 6umol of H2folate/min under the specified conditions. For the kinetic studies a variety of buffers was used, details of which are given in the Results and Discussion section; all assays were carried out at 25°C.…”

Section: Enzyme Assaymentioning

confidence: 99%

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Dann¹,

Ostler²,

Bjur³

et al. 1976

Biochemical Journal

150

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Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5g of enzyme is obtained from a 400 litre culture. The purification procedure, involving polyethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. ofapprox. 17900 and a turnover number of4s-(5OmM-triethanolamine/400mM-KCI, pH7.2, 25°C) with dihydrofolate and NADPH as substrates.The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.361uM-dihydrofolate; 0.78 uM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 x 106M-1; NADPH, >108M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is responsible for maintaining the intracellular pool of tetrahydrofolate by reducing dihydrofolate (arising either by biosynthesis de novo or by the action of thymidylate synthetase on 5,10-methylenetetrahydrofolate) to tetrahydrofolate (Blakley, 1969). This enzyme is of considerable pharmacological interest as the site of action of a group of powerful chemotherapeutic agents, the 'anti-folates', which includes methotrexate, trimethoprim and pyrimethamine (Blakley, 1969;Baker, 1967).We are undertaking a detailed study of the binding of substrates, coenzymes and inhibitors to the dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei, principally by highresolution n.m.r. (nuclear-magnetic-resonance) spec-* Present address:

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“…This inter pretation is supported by following observa tions: (a) biodegradation of folic acid is brought about in the reduced level (12,23,24), (b) the formyldihydropteridine is readily formed stoichiometrically from dihydrof olic acid, but not from tetrahydrof olio acid, under the physiological conditions by action of molecular oxygen (11,25), (c) tetrahydrofolate coenzymes can be stabilized against oxidation by such sub stitutions at the position 5 as 5, 10-methylene, 5-formyl, 5, 10-methylenyl and 5-methyl adducts (29), (d) the formyldihydropteridine is relatively stable to oxidation in neutral solution, differed from other 6-alkylated dihydropteridines (11), (e) as confirmed in the present work the formyldihydropteridine can be converted en ymatically to dihydropteroic acid, and (f) inhibition of E.…”

Section: Resultsmentioning

confidence: 82%

Enzymatic Conversion of 2-Amino-4-hydroxy-6-formyl-7, 8-dihydropteridine to 2-Amino-4-hydroxy-6-hydroxymethyl-7, 8-dihydropteridine by Cell-free Extracts of Escherichia coli B

Mitsuda

Suzuki

1971

J. Vitaminol

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Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase

Cited by 243 publications

References 20 publications

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Enzymatic Conversion of 2-Amino-4-hydroxy-6-formyl-7, 8-dihydropteridine to 2-Amino-4-hydroxy-6-hydroxymethyl-7, 8-dihydropteridine by Cell-free Extracts of Escherichia coli B

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