NADPH-cytochrome P-450 reductase from untreated rat testicular and liver microsomes: isolation and comparison.
NADPH-cytochrome P-450 reductase (EC 1.6.2.4), the enzyme involved in progesterone 17-hydroxylation, was purified to apparent homogeneity by detergent solubilization of the microsomal fractions of liver and testis from untreated rats. The enzymes from these two tissues were then compared with regard to several parameters. The liver and testicular reductases have the same subunit mol wt (79,000), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and have similar specific activity for cytochrome c reduction (48.1 and 58.2 mumol min-1 mg-1, respectively). The absolute flavin absorption spectra of liver and testicular reductase were very similar. The spectra of native, reduced, and oxidized reductases were characteristic of flavoprotein with two flavin groups. The Km values for NADPH during cytochrome reduction were shown to be the same, within experimental error (5.29 +/- 0.46 microM-1 for liver and 4.37 +/- 0.53 microM-1 for testis). Monospecific antiserum was prepared against both rat liver and testicular reductases. There were no antigenic differences demonstrated by immunological tests using either antibody preparation. Antiliver reductase antiserum was shown to inhibit testicular microsomal progesterone 17-hydroxylase activity by 70%. Testicular cytochrome c reductase activity was inhibited by 94%, and liver cytochrome c reductase activity was inhibited by 85%. Peptide maps of both forms of reductase isolated from immunoprecipitates showed very similar patterns after enzymatic proteolysis. These results indicate that microsomal NADPH-cytochrome P-450 reductase involved in steroid hydroxylations in untreated rat liver and testis could not be distinguished from each other by these methods and, therefore, are probably the same protein.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D000906:Antibodies; D000939:Epitopes; D009251:NADPH-Ferrihemoprotein Reductase