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Male sex hormones [dihydrotestosterone (DTS), and testosterone] and progesterone, when added to the isolated rat liver mitochondria before or after some protonophores, lower the respiration rate and increase the delta psi level, i.e., reverse the protonophore-induced uncoupling. Such a recoupling ability shows specific structural requirements correlating with hormonal activity of steroids studied. For instance, epiandrosterone, a DTS isomer of very low hormonal activity, and deoxycorticosterone, differing from progesterone by additional OH-group and possessing quite different hormonal activity, as well as female sex hormones (estron and estradiol) show no recoupling effect. Like 6-ketocholestanol (kCh), male sex hormones and progesterone recouple mitochondria uncoupled by low concentrations of SF6847, FCCP and CCCP, but not by high concentration of these uncouplers or by any concentration of DNP, palmitate and gramicidin. In contrast to recoupling by kCh, hormonal recoupling requires addition of serum albumin and is inhibited by low concentrations of palmitate. Recoupling can also be shown on the heart and skeletal muscle mitochondria, being absent from the heart muscle submitochondrial particles, the bacterial chromatophores and the cytochrome oxidase proteoliposomes. In mitochondria it does not depend upon the oxidation substrate used (succinate or PMS + ascorbate were tested). Pronounced seasonal effect upon the DTS recoupling degree was revealed. The recoupling is maximal in January, February and from June to November, being minimal in the spring months and in December. In spring, the in vivo administration of thyroxine, di- or triiodothyronine improves the recoupling ability of DTS. 2 x 10 - 6 M. Thyroxine, when added in vitro, does not affect energy coupling if SF6847 was absent. In the presence of small amounts of SF6847, thyroxine stimulates the uncoupling in a DTS-sensitive fashion, di- and triiodothyronines being less effective. Addition of thyroxine to azide-inhibited mitochondria (oligomycin is present) stimulates respiration and normalizes the delta psi level. In this system, triiodothyronine is much less effective, whereas diiodothyronine is not effective at all. In the intact cells (thymocytes and the Krebs-II cells were tested), DTS lowers the respiration rate stimulated by low concentrations of SF6846 or FCCP. In this case, serum albumin is not required. It is suggested that recoupling effects of male sex hormones and progesterone are involved in their anabolic action just as uncoupling takes part in the catabolic activity of thyroid hormones.

The effect of 6-ketocholestanol (kCh) on various natural and reconstituted membrane systems has been studied. 6-ketocholestanol (5 alpha-Cholestan-3 beta-ol-6-one), a compound increasing the membrane dipole potential, completely prevents or reverses the uncoupling action of low concentrations of the most potent artificial protonophore SF6847. This effect can be shown in the rat liver and heart muscle mitochondria, in the intact lymphocytes, in the Rhodobacter sphaeroides chromatophores, and in proteoliposomes with the heart muscle or Rh. sphaeroides cytochrome oxidase. The recoupling effect of kCh disappears within a few minutes after the kCh addition and cannot be observed at all at high SF6847 concentrations. Almost complete recoupling is also shown with FCCP, CCCP, CCP and platanetin. With 2,4-dinitrophenol, fatty acids and gramicidin, kCh is ineffective. With TTFB, PCP, dicoumarol, and zearalenone, low kCh concentrations are ineffective, whereas its high concentrations recouple but partially. The kCh recoupling is more pronounced in mitochondria, lymphocytes and proteoliposomes than in chromatophores. On the other hand, mitochondria, lymphocytes and proteoliposomes are much more sensitive to SF6847 than chromatophores. A measurable lowering of the electric resistance of a planar bilayer phospholipid membrane (BLM) are shown to occur at SF6847 concentrations which are even higher than in chromatophores. In BLMs, kCh not only fails to reverse the effect of SF6847, but even enhances the conductivity increase caused by this uncoupler. It is assumed that action of low concentrations of the SF6847-like uncouplers on coupling membranes involves cytochrome oxidase and perhaps some other membrane protein(s) as well. This involvement is inhibited by the asymmetric increase in the membrane dipole potential, caused by incorporation of kCh to the outer leaflet of the membrane.

The synthesis of inorganic pyrophosphate by the mitochondria of Endomyces magnusii was found to be coupled with respiration. The mitochondria of E. magnusii can utilize the energy of the phosphoanhydride bond of inorganic pyrophosphate for the synthesis of ATP. The study with inhibitors suggests the participation of inorganic pyrophosphatase and ATPase in this process.

A sensitive colorimetric method for the assay of inorganic pyrophosphate with excess of orthophosphate is described. The principle of this method lies in the formation of phosphomolybdate and PPi-molybdate complexes with subsequent extraction of the phosphomolybdate complex by organic solvents and reduction of the PPi-molybdate complex by dithiothreitol and Eikonogen. The sensitivity of the method was from 5 to 120 nmol of PPi in a 2.0-ml sample.

The effect of temperature on the hydrolase activity of mitochondrial pyrophosphatases, i.e. soluble (PPase I) and membrane (PPase II), has been studied. In contrast to the soluble species, the membrane form has inflexions in the Arrhenius curves. However, after lipidization of PPase I by various phospholipids, the curves also acquire inflexion points, which prove similar or identical with the phase separation points of the lipids used. The closeness of the inflexion points of PPase II, containing phosphatidylcholine, and PPase I lipidized by mitochondria phosphatidylcholine, to the phase separation points of this lipid indicates that the inflexions on the PPase II curves should be ascribed to this phospholipid. It has been shown that the hydrolysis of PPi by SMP is affected by the cooperative rearrangements of the entire lipid component of the membrane rather than by the change of the phase state of PPase II phosphatidylcholine. Reconstitution experiments on the PPi synthesis system have shown that after lipidization PPase I is able to incorporate into SMP and become a coupling factor for respiration and PPi synthesis, like PPase II.