AVIAN KIDNEY MITOCHONDRIAL HEMEPROTEIN-P-4501-ALPHA - ISOLATION, CHARACTERIZATION AND NADPH-FERREDOXIN REDUCTASE-DEPENDENT ACTIVITY

We describe the isolation of cytochrome P-4501α 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-4501α 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-4501α traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57 000. The absolute spectrum of the P-4501α(Fe3+) form gave a λmax at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501α 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-4501α catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3β-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. © 1990.