Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes

Biochemistry. 1977 Aug 9;16(16):3566-72. doi: 10.1021/bi00635a010.


A combination of affinity column chromatography and preparative gel electrophoresis has been used to purify to homogeneity the two isozymes of dihydrofolate reductase from a trimethoprim-resistant strain of Escherichia coli B (RT 500). These enzyme forms are noninterconvertible and are present in crude cell lysates, but other electrophoretic species can be generated durng purification if sulfhydryl-protecting agents, such as dithiothreitol, are not present. The two isozymes, numbered form 1 and form 2 with respect to their decreasing electrophoretic mobilities, have similar molecular weights (18 500), molecular radii (21 A), and apparent Km values for reduced nico inamide adenin- dinucleotide (NADH) and NADH phosphate (NADPH). Both forms contain 2 mol of sulfhydryl/mol of enzyme which can be oxidized to intramolecular disulfide bonds. However, 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.

MeSH terms

  • Escherichia coli / enzymology*
  • Hydrogen-Ion Concentration
  • Isoenzymes / isolation & purification*
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Weight
  • Tetrahydrofolate Dehydrogenase / isolation & purification*
  • Tetrahydrofolate Dehydrogenase / metabolism


  • Isoenzymes
  • Tetrahydrofolate Dehydrogenase