Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase.
A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000276:immunology / Q000302:isolation & purification*; D005288:Ferredoxins / Q000378:metabolism; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D013552:Swine
D005288:Ferredoxins; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases