Purification and characterization of dihydrofolate reductase from methotrexate-resistant human lymphoblastoid cells

Abstract

Dihydrofolate reductase was isolated from methotrexate-resistant WIL2 human lymphoblastoid cells. This subline produces .apprx. 150 times more enzyme than the parental drug-sensitive line. The reductase was purified to homogeneity by methotrexate affinity chromatography, gel filtration and preparative isoelectric focusing in a yield of 65%. The enzyme has a pI [isoelectric point] = 7.7 and a MW of .apprx. 22,000. The amino-terminal 27 amino acid residues were determined, revealing the location of the single cysteine residue at position 6. Reaction of this cysteine with p-(hydroxymercuri)benzoate in the presence of NADPH results in a 5-fold increase in enzyme activity. Other agents including KCl, urea and thiourea also cause enzyme activation. The Km values for NADPH and dihydrofolate are 0.25 and 0.036 .mu.M, respectively. Mercurial activation of the enzyme results in a 27-fold increase in the Km for NADPH and a 25-fold increase in the Km for dihydrofolate.