Escherichia coli dihydrofolate reductase: isolation and characterization of two isozymes.
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.
D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D013762:Tetrahydrofolate Dehydrogenase