Avian kidney mitochondrial hemeprotein P-4501 alpha: isolation, characterization and NADPH-ferredoxin reductase-dependent activity.
We describe the isolation of cytochrome P-4501 alpha from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-4501 alpha could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-4501 alpha traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57,000. The absolute spectrum of the P-4501 alpha (Fe3+) form gave a lambda max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501 alpha which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275-8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933-3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-4501 alpha catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3 beta-ol at the C-1 position exclusively with a turnover number of 0.03 min-1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.
Mandel
M L
ML
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53225.
Swartz
S J
SJ
Ghazarian
J G
JG
D000818:Animals; D002112:Calcifediol / Q000378:metabolism; D002117:Calcitriol / Q000378:metabolism; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D006900:Hydroxylation; D007668:Kidney / Q000648:ultrastructure*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D013053:Spectrophotometry
D003577:Cytochrome P-450 Enzyme System; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D002117:Calcitriol; D002112:Calcifediol