Multiple Forms of Dihydrofolate Reductase in Cultured Mammalian Cells

Abstract

<p> Dihydrofolate reductase from a subline of the L1210 lymphoma was purified by affinity chromatography using substituted Sepharose -4B to which was coupled methotrexate, a specific, tight binding inhibitor of the enzyme. The purified enzyme was subjected to disc gel electrophoresis at pH 8.5. At least two bands of activity were detected on the gel by the formation of a reduced formazan. Their ratios were dependent on enzyme concentration. Similar bands were found in the presence of EDTA (10^-6M), 4M and SM urea. When a substrate, NADPH (5xl0^-5M), was added to the buffers used in electrophoresis, three bands of enzyme activity were present in a fixed ratio which was independent of enzyme concentration. Protein bands showed a different but constant ratio. When folate replaced dihydrofolate as substrate in the assay mixture, the bands of activity corresponded at high concentrations of the enzyme. When activity was detected in the presence of an increasing concentration of methotrexate, different inhibition of the bands resulted. Preliminary experiments with crude extracts of the same subline gave activity profiles with multiple peaks.</p>