Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone-induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membranelmass protein and a change in the intrinsic permeability of the phospholipid bilayer.Mitochondria isolated from the livers of hypothyroid animals ('hypothyroid mitochondria') have respiration rates that are lower than euthyroid controls, and mitochondria isolated from the livers of hyperthyroid animals ('hyperthyroid mitochondria') have respiration rates that are higher than euthyroid controls [1,2]. These changes in respiration rate are also seen in isolated hepatocytes [3], showing that they are not artefacts of isolation of the subcellular organelles. They are thought to be an important part of the changes in metabolic rate and heat production controlled by thyroid hormones There are two separate effects of thyroid hormone levels on respiration rate of isolated mitochondria: one is important in state-3 respiration, in which ADP is rapidly phosphorylated to ATP; the other is dominant in conditions in which there is little or no ATP synthesis [7, 81. The main effect on state-3 respiration is a stimulation by thyroid hormones of the reactions involved in the phosphorylation of ADP and the export of ATP; probably at the ad...

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The supply of electrons for the hydroxylation reactions associated with cholesterol side-chain cleavage has been studied in intact porcine luteal mitochondria. The work was based on an assay of the NADPH-dependent side-chain-cleavage reaction, the activity of the mitochondria1 enzyme complex being expressed as the percentage of [4-14C]cholesterol converted to the products, [4-14C]pregnenolone and [4-14C]progesterone, in 1 h. The reaction could best be supported by intermediates of the tricarboxylic acid cycle, although NADPH, and to a lesser degree NADH, would also support some activity.Investigations of the ADP:O ratios of the substrates which could support cholesterol side-chain cleavage showed that they were lower than the ADP:O ratios found for the same substrates in liver mitochondria from the same animal; respiratory-control ratios were also lower in the ovarian preparation. In the adrenal cortex, another steroid-hydroxylating tissue, similar results obtained by other workers have been interpreted as indicating that the two oxidative chains, the first terminating in cytochrome oxidase and the second in cytochrome P450, are connected by an energy-dependent transhydrogenase. In the ovary this explanation may be true for succinate, but it does not satisfy the results obtained with substrates which reduce NAD(P)+. Although spectrophotometric assays showed that there was some energy-linked transhydrogenase, as well as non-energy-linked transhydrogenase activity in ovarian mitochondria, all the evidence obtained by investigating cholesterol side-chain cleavage supported by NADH and the NAD+-specific 2-oxoglutarate dehydrogenase indicated that in ovary hydrogen transfer from NADH to the cholesterol-hydroxylation site was not dependent on energy. I n contrast, inhibitors of NADH oxidase markedly stimulated NADH-supported side-chain cleavage. Energy was required for the transfer of electrons from succinate to the site of cholesterol side-chain cleavage.Mitochondria from ovarian tissue possess in addition to the normal respiratory cytochrome system [i] a second electron-transport chain concerned with steroid-hydroxylation reactions [l-31 and in particular, with cholesterol side-chain cleavage. This latter electron-transport chain, like that associated with steroid hydroxylation in adrenal-cortex mitochondria [4,5] Trivial Names. Cholesterol, 5-cholesten-3/?-01; deoxycorticosterone, 21-hydroxy-4-pregnene-3,20-dione; pregnenolone, 3/?-hydroxypregn-5-en-20-one ; progesterone, 4-pregnene-3,2O-dione.

Cytochrome P-45OS,,, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The K,,, for cholesterol of purified luteal cytochrome P-45OS,, incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in K,,,. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450,,,.Cytochrome P-450,,, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues is located in the inner mitochondrial membrane, on the matrix side [l, 21 and appears to be identical in bovine adrenal cortex and bovine corpus luteum [3]. The cytochrome has its cholesterol binding site in the hydrophobic phospholipid milieu and has a ferredoxin binding site exposed to the matrix [3 -81. Ferredoxin, a one electron carrier, shuttles between ferredoxin reductase and cytochrome P-450,,, supplying the six electrons required to convert cholesterol to pregnenolone [8]. Ferredoxin and ferredoxin reductase also appear identical between the corpus luteum and adrenal cortex [9]. Cytochrome P-450,,, can easily be removed from the membrane with detergents but incorporation of the purified cytochrome into a membrane or detergent micelle is essential before it can catalyse the conversion of the water insoluble cholesterol to pregnenolone.Incorporation of purified cytochrome P-450,,, into artificial phospholipid vesicles has revealed that cholesterol binding to the cytochrome is dependent on the phospholipid composition of the membrane [3 -81. Cardiolopin in particular appears to be a potent stimulator of cholesterol binding to the cytochrome [3,. It has been reported that in the presence of cardiolipin the K,,, for cholesterol expressed as mol cholesterol per mol of phospholipid is as low as 0.07 [6]. However, these studies were done with vesicles prepared from commercial phospholipids, the compositions of which do not reflect closely the composition of the inner mitochondrial Correspondence to