Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes

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Abstract
A combination of affinity column chromatography and preparative gel electrophoresis was used to purify to homogeneity the 2 isozymes of dihydrofolate reductase from a trimethoprim-resistant strain of E. coli B (RT 500). These enzyme forms are noninterconvertible and are present in crude cell lysates, but other electrophoretic species can be generated during purification if SH-protecting agents, such as dithiothreitol, are not present. The 2 isozymes, numbered form 1 and form 2 with respect to their decreasing electrophoretic mobilities, have similar MW (18,500), molecular radii (21 .ANG.) and apparent Km values for NADH and NADPH. Both forms contain 2 mol of SH/mol of enzyme which can be oxidized to intramolecular disulfide bonds. Forms 1 and 2 differ physically in their electrophoretic mobility and isoelectric point and kinetically in their pH-activity profile, specific activity, Km for dihydrofolate and their affinity toward a number of inhibitors.