Submitochondrial Distribution of Components of the Steroid 11β-Hydroxylase and Cholesterol Sidechain-Cleaving Enzyme Systems in Hog Adrenal Cortex

Abstract

1. Osmotic shock followed by discontinuous Ficoll gradient centrifugation was applied to hog adrenocortical mitochondria to localize components of the steroid 11β-hydroxylase [EC 1. 14. 1.6] system and of the cholesterol sidechain-cleaving enzyme system in submitochondrial fractions.
2. The supernatant obtained by centrifugation at 17, 000×g after the osmotic shock at 0°C in hypotonic phosphate buffer, contained high malate dehydrogenase [EC 1. 1. 1. 37] activity. Neither the steroid 11β-hydroxylase system nor the cholesterol sidechain-cleaving enzyme system was found in this fraction.
3. Of the four fractions obtained by discontinuous Ficoll Gradient centrifugation after osmotic shock, the uppermost fraction was judged to contain the outer membranes of mitochondria from its contents of monoamine oxidase [EC 1. 4. 3. 4], Amytal-insensi-tive NADH cytochrome c reductase and a cytochrome tentatively identified as cyto-chrome b₅. A significant amount of antimycin A-insensitive NADPH cytochrome c reductase was also found in this fraction.
4. The pellet and two other membranous fractions obtained by discontinuous Ficoll gradient centrifugation seemed to contain materials derived from the inner membranes of mitochondria. NADPH diaphorase [EC 1. 6. 99. 1], cytochrome P-450, steroid 11β-hydroxylase and enzymes which participate in the reaction cleaving the cholesterol sidechain, i.e., 20α-hydroxylase, 22-hydroxylase and C20, 22-lyase, could be localized in the inner membrane but not in the outer membrane. Adrenodoxin seemed to leak out of these enzyme complexes of steroid hydroxylation during preparation of submitochondrial fractions.