STEROID HYDROXYLATIONS IN RAT ADRENAL MITOCHONDRIA .I. SOME PROPERTIES OF C-18 AND 11-BETA HYDROXYLATIONS IN TIGHTLY COUPLED MITOCHONDRIA

Mitochondrial preparations have been isolated from cortex fragments, adrenal capsules, and intact rat adrenal glands and shown to have high ADP O and respiratory control ratios with all substrates tested. It was necessary to exclude Mg2+ because of an apparently nonmitochondrial Mg2+-stimulated ATPase. These preparations also gave high rates of both the C-18 and 11-β steroid hydroxylation. Steroid hydroxylation with β-hydroxybutyrate or α-ketoglutarate plus malonate was energy-dependent. With the latter substrate, conditions were found which made steroid hydroxylation dependent on both the enzymes of oxidative phosphorylation and on energy conserved at the substrate-linked phosphorylation site. Isocitrate supported steroid hydroxylation by a nonenergy-dependent pathway, presumably via the active NADP-linked isocitric dehydrogenase present in these preparations. Steroid hydroxylation supported by malate was decreased by KCN and uncouplers of oxidative phosphorylation. With malate plus either inorganic phosphate or arsenate as substrate, however, an additional KCN and uncoupler insensitive pathway for corticosterone production became evident. In this latter situation, pyruvate accumulated suggesting the activation of a malic enzyme-like activity by phosphate or arsenate in the intact adrenal mitochondria. It is concluded that rat adrenal mitochondria have efficient energy-conserving reactions, and that steroid hydroxylation may proceed by energy-dependent, nonenergy-dependent, or by both pathways simultaneously depending on the substrate utilized. © 1969.