Isolation and characterization of the multiple forms of dihydrofolate reductase from methotrexate-resistant hamster cells.

Dihydrofolate reductase, isolated from baby hamster kidney cells by affinity chromatography, gave multiple bands on analytical gel electrophoresis and electrofocusing. The number of bands could be reduced to two by forming a complex with the enzyme inhibitor, methotrexate. The two basic enzyme forms, called Form I and Form II, were obtained in large quantities by preparative disc electrophoresis and analyzed for complex formation, aggregation, conformation, composition, catalytic activity, and interconversion. Each form produced stable and electrophoretically distinguishable complexes with substrate, substrate analogues, cofactor, and oxidized cofactor. The complex formation with the cofactors was accompanied by a change in the conformation. Form I and Form II differed in charge, conformation or size, specific activity, and pH optimum. They were not themselves cofactor complexes, nor was one of them a multimer or aggregate of the other. No interconversion could be shown. Analysis of the tryptic and chymotryptic digests of the two forms failed to show a difference in their amino acid sequence, but a small change in the sequence or certain physical or chemical modifications of the protein would not have been detected. We conclude that the multiple bands of dihydrofolate reductase are due to the presence of two closely related enzyme forms which independently produce stable and distinguishable complexes with the substrate, the cofactor, and the oxidized cofactor.