R plasmid dihydrofolate reductase with subunit structure.
Dihydrofolate reductase, specified by the type II plasmid of a trimethoprim-resistant Escherichia coli, was purified 40-fold to homogeneity using a combination of gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography. The final product shows a single protein band on polyacrylamide gel electrophoresis and has a specific activity of 1.0 unit/mg. The molecular weight of the purified enzyme is 36,000 as determined both by gel filtration and Ferguson analysis of polyacrylamide gel electrophoresis. In contrast, a single polypeptide with a molecular weight of 8,500 was observed on sodium dodecyl sulfate-gel electrophoresis. These experiments suggest that, unlike any bacteria or vertebrate dihydrofolate reductase previously examined, the type II R plasmid reductase is a tetramer composed of four identical subunits. A partial amino acid sequence determination shows no heterogeneity of the subunits and also no clear homology with any reductase sequence previously reported.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D014170:Transformation, Genetic; D014295:Trimethoprim / Q000494:pharmacology
D046911:Macromolecular Substances; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase