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Dihydrofolate Reductase from the L1210 R Murine Lymphoma

1967; Elsevier BV; Volume: 242; Issue: 20 Linguagem: Inglês

10.1016/s0021-9258(18)99523-2

1083-351X

Brian L. Hillcoat, John P. Perkins, Joseph R. Bertino,

Bipolar Disorder and Treatment

Abstract Quenching of fluorescence of highly purified dihydrofolate reductase from a methotrexate-resistant subline of the L1210 lymphoma by substrates and inhibitors was used to determine dissociation constants between pH 5 and pH 9. At pH 7.0, the dissociation constant of dihydrofolate from the enzyme-dihydrofolate complex was 1.2 x 10-7 m, 10 times greater than that of NADPH and several times less than that of folate at the same pH. Dihydrofolate, folate, or NADPH binding to the enzyme in binary complex did not vary significantly with pH, except for a 5-fold decrease in the binding of folate at pH 9.0. Triamterene (2,4,7-triamino-6-phenylpteridine) binding to the enzyme in binary complex showed a marked decrease above pH 7.0 and was not detectable at pH 9.0. The binding of NADPH in ternary complex of enzyme, triamterene, and NADPH was 10 times greater than that in binary complex of enzyme and NADPH, and did not vary significantly with pH except for a 4-fold decrease at pH 9.0, probably related to the decreased binding of triamterene at this pH. Pyrimidine inhibitors which possess a hydrophobic side chain were bound more tightly in ternary than in binary complex, whereas those with a p-aminobenzoylglutamic acid side chain were not. It is suggested that hydrophobic bonding may be enhanced in ternary complex because of a possible conformational change in enzyme when combined with NADPH.