Leonard A. Sauer

Steroid hydroxylations in rat adrenal mitochondria: I. Some properties of the C-18 and 11-β hydroxylations in tightly coupled mitochondria☆

Abstract 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.

An NAD- and NADP-dependent malic enzyme with regulatory properties in rat liver and adrenal cortex mitochondrial fractions

Abstract The NAD- and NADP-dependent malic enzymes from rat liver and adrenal mitochondrial fractions were separated and partially purified by gel filtration on Sepharose 6B. Two activity peaks were observed. The first contained a malic enzyme capable of reducing either NAD or NADP. This enzyme showed sigmoid kinetics in plots of activity versus the malate concentration. Succinate was an allosteric activator and ATP was a competitive inhibitor of malate. The second peak showed hyperbolic kinetics in plots of activity versus the malate concentration and was unaffected by either succinate or ATP. The relative activities of the two malic enzymes were quite constant in the adrenal mitochondrial fractions. In the liver mitochondrial fractions, the activity of the first peak varied and was sometimes absent while the activity of the second peak was quite constant. The kinetic properties of the first malic enzyme implicate it as an important regulator of malate oxidation.

Mitochondrial NAD‐dependent malic enzyme: A new regulatory enzyme

1. Introduction Electrophoretic evidence for multimolecular forms of malic enzyme (EC 1.1.1.40, NADP-malic dehydro- genase, decarboxylating) was reported by Henderson in 1966 [ 11. Subsequent purification and assay by spectrophotometric and other methods revealed that malic enzyme isoenzymes were present in cytosol and mitochondrial fractions derived from several different animal tissues [2-61. Of particular importance were the recent findings of Frenkel [7] that the mitochon- drial isoenzyme has allosteric properties, and that succinate was a positive modulator. Here, we demon- strate that calf adrenal cortex mitochondria contain an NAD-dependent malic enzyme (EC 1.1.1.39) with regulatory properties. Fumarate was a positive and ATP a negative modulator. The enzyme also reduced NADP but only at about one-quarter of the rate of NAD reduction. These kinetic properties suggest that this enzyme is an important regulator of the rate of malate oxidation. 2. Materials and methods Calf adrenal glands were obtained from the local

Steroid hydroxylations in rat adrenal mitochondria. II. Competition between energy-linked transhydrogenase-dependent steroid hydroxylation and oxidative phosphorylation for high-energy intermediates and NADH.

Abstract In the presence of 30 μ m deoxycorticosterone (DOC), 10 m m alpha-ketoglutarate + 10 m m malonate, ADP, and inorganic phosphate, rat adrenal mitochondria supported nearly maximal rates of both oxidative phosphorylation and steroid hydroxylation. These rapid reactions were able to occur simultaneously because this steroid concentration only slightly inhibited the electron transport of oxidative phosphorylation. During the oxidation of alpha-ketoglutarate or β-OHbutyrate, however, ATP formation by oxidative phosphorylation was inhibited competitively by steroid hydroxylation supported by the energy-linked transhydrogenase. ATP formation during β-OHbutyrate oxidation was inhibited to a greater extent by steroid hydroxylation than that supported by alpha-ketoglutarate oxidation. That this inhibition was due to a competition was shown by the fact that metopirone, an inhibitor of 11-β hydroxylation, inhibited DOC consumption and increased ATP formation by oxidative phosphorylation to control levels. The stoichiometry of the competitive reactions suggests that at high rates of NADH formation one high-energy intermediate is consumed for each steroid molecule hydroxylated. At low rates of NADH formation four high-energy intermediates are consumed for each molecule of DOC consumed. These results were interpreted to mean that the energy-linked transhydrogenase utilizes high-energy intermediates and NADH from the pools available to oxidative phosphorylation. When the rate of intramitochondrial NADH formation is low, the energy-linked transhydrogenase appears to have preferential access to the NADH.

The submicrosomal distribution of radioactive proteins released by puromycin from the bound ribosomes of rat liver microsomes labelled in vitro

Abstract 1. The puromycin-mediated release of labelled-nascent proteins from bound ribosomes was examined in rat liver microsomes labelled in vitro with [ 14 C]leucine. Subfractions were prepared from labelled control and puromycin-treated microsomes either by treatment with 0.25 % sodium deoxycholate or by sonication. 2. When the microsomes were fractionated by sodium deoxycholate, the distribution of the acid-insoluble radioactivity among the microsomal subfractions indicated that the puromycin-mediated release could be directed either toward the microsomal membrane or toward the intravesicular space. An unequivocal interpretation is not possible because of subfraction contamination and protein binding phenomena. 3. However, the distribution of radioactivity following gel filtration of deoxycholate-solubilized labelled microsomal membranes, or dispersion of the labelled microsomes by sonication, suggest that the direction of the puromycin-mediated release is toward the microsomal membrane rather than toward the intravesicular lumen.

On the intracellular localization of the 3beta-hydroxysteroid dehydrogenase isomerase in the rat adrenal cortex.

Summary In this study we have compared some properties of the microsomal and mitochondrial 3β-hydroxysteroid dehydrogenase/isomerase (HSD) from the rat adrenal cortex. The following major differences were noted: (i) the microsomal enzyme required exogenous NAD while the mitochondrial enzyme was active in the absence of added NAD. (ii) The mitochondrial enzyme was inhibited by a combination of rotenone, KCN, and citric acid cycle substrates. These agents did not influence the activity of the microsomal enzyme, (iii) The ratio of the specific activities of HSD to the 21-hydroxylase (a microsomal marker enzyme) was greater in the mitochondria than in the microsomes. We conclude that HSD is present in the inner mitochondrial membrane and, therefore, that the enzyme has a dual mitochondrial and microsomal localization in the rat adrenal cortex. The authors wish to thank Drs. P. J. Mulrow and S. Y. Tan for several helpful discussions during the course of this study.

Steroid hydroxylations in rat adrenal mitochondria: IV. An inhibition of NADH oxidase and succinate-supported deoxycorticosterone hydroxylation by steroid and rotenone☆

Abstract The effects of steroid and rotenone on succinate-supported deoxycortieosterone hydroxylation were compared. In intact rat adrenal mitochondria, the rate of deoxycortieosterone hydroxylation decreased as the concentration of the steroid substrate or the major steroid product (corticosterone) or rotenone was increased. Steroid had no effect on either succinate-supported oxidative phosphorylation or on deoxycorticosterone hydroxylation supported by NADPH plus calcium ions. In submitochondrial particles, NADPH formation by the energy-linked transhydrogenase was only slightly inhibited by steroid. NADH oxidase, however, was markedly inhibited by deoxycortieosterone, corticosterone or rotenone suggesting that steroid (like rotenone) decreased the rate of succinate-supported steroid hydroxylation by specifically inhibiting reversed electron transport. Further support for this interpretation was found in the inhibition by either steroid or rotenone of the ATP-dependent, succinate-supported hydroxylation of deoxycortieosterone in KCN-inhibited mitochondria. It is concluded that the deleterious effect of steroid on succinate-supported steroid hydroxylation is due to an inhibition of reversed electron transport in the region of the first phosphorylation site. The steroid, rotenone and antimycin sensitivity of the NADH oxidase of rat adrenal submitochondrial particles differed depending on the method of assay. When measured by NADH disappearance, NADH oxidase activity was sensitive to all three inhibitors and KCN. When measured by oxygen consumption, NADH oxidase activity was sensitive to KCN but only slightly sensitive to steroid, rotenone or antimycin. Rat adrenal submitochondrial preparations were found to contain an active rotenone-and antimycin-insensitive NADH-cytochrome c reductase in addition to the typical inhibitor-sensitive NADH oxidase. Exogenous cytochrome c converted the rotenone or antimycin-sensitive NADH oxidase to an inhibitor-insensitive NADH oxidase. However, sensitivity to KCN remained. The results indicate that a shuttle of cytochrome c between the inhibitor-insensitive NADH cytochrome c reductase and the inhibitor-sensitive NADH oxidase may explain the rotenone- and antimycin-insensitive NADH oxidase activity of adrenal cortex submitochondrial preparations.

Steroid hydroxylations in rat adrenal mitochondria III. The ATP-steroid-oxygen stoichiometry of ATP-dependent steroid hydroxylation

Abstract The energy—steroid—oxygen stoichiometry of ATP-dependent 11-deoxy-corticosterone hydroxylation was measured in tightly coupled rat adrenal mitochondria in the presence of KCN and rotenone. The substrate was β-hydroxybutyrate. The results indicate that one molecule of steroid is hydroxylated for each molecule of ATP utilized. It is concluded that in intact rat adrenal mitochondria the stoichiometry of the energy-linked transhydrogenase is one NADPH formed for each high-energy intermediate of oxidative phosphorylation consumed.