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Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin alpha (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin alpha (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H(2)O(2), catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.

Earlier we reported that some thyroid and steroid hormones and also 6-ketocholestanol used in micromolar concentrations modulated the effects of protonophoric uncouplers on isolated mitochondria (Starkov et al. (1997) Biochim. Biophys. Acta, 1318, 173-183). In the present study we investigated the effects of a thyroid hormone, thyroxine, on energy coupling of intact rat thymus lymphocytes and mitochondria isolated from these cells. The resting (oligomycin-inhibited) respiration of the isolated intact lymphocytes was stimulated by the addition of protonophoric uncouplers 2,4-DNP, FCCP, or SF6847. Subsequent addition of micromolar concentrations of thyroxin decreased the rate of uncoupler-stimulated respiration and partially reversed uncoupler-induced decrease of membrane potential (DeltaPsi). In experiments with mitochondria isolated from thymus lymphocytes the re-coupling effect of thyroxine was not observed. In this case thyroxine did not influence mitochondrial respiration stimulated with 2,4-DNP, but did potentiate the stimulation of respiration and DeltaPsi decrease induced with another uncoupler, SF6847. The data are discussed in terms of a hypothesis that aromatic uncouplers are transported into the cell by the thyroxine carrier of the plasma membrane.

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.

A convenient procedure is proposed for extracting mitochondrial proteolipids using a single phase mixture chloroform-methanol-water (1:2:0.8 v/v) with subsequent separation of the phases. The proteolipids were concentrated at the interface between the phases and thus purified from the bulk of the phospholipids. It was found that the mitochondrial proteolipids represent stable complexes of phospholipids with some low molecular weight proteins (M(r) = 7-18 kDa). The latter are destroyed at acid pH values. The phospholipid/protein ratio was found to be equal to 6 (assuming the molecular masses of the proteins and phospholipids to be equal to 10 and 0.8 kDa, respectively). The phospholipid composition of the tightly bound proteolipids thus obtained did not differ from that of the mitochondrial phospholipids. Using 31P-NMR, nonbilayer structures were found to arise from proteolipid reconstitution into multibilayer liposomes.

The effects of lipids on the activity of soluble and membrane-bound pyrophosphatase from beef heart mitochondria were studied. An addition of total mitochondrial lipid, phosphatidyl choline, phosphatidyl ethanolamine or cardiolipin resulted in stimulation of the enzymatic activity and an increase in thermal stability of the soluble enzyme. The maximal activating effect was exerted by the total mitochondrial lipid and phosphatidyl choline. The electrophoretic data suggest that phosphatidyl choline is a component of membrane pyrophosphatase. Preincubation of the soluble enzyme with phosphatidyl choline converted the enzyme into a membrane form, which is capable to carry out the energy-dependent synthesis of PPi in submitochondrial particles.

It was demonstrated previously that mitochondria of higher and lower eukaryotes can synthesize, in the course of oxidative phosphorylation, not only ATP but also inorganic pyrophosphate (PPi). Two PPases were isolated from bovine heart mitochondria (soluble--PPase I and membrane--PPase II). Coupling PPase II, in contrast to PPase I, contains phosphatidyl choline, but PPase I is lipidized readily in the presence of different phospholipids. Reconstitution experiments of the PPi synthesis system have shown that after lipidization PPase I is able to incorporate into submitochondrial particles (SMP) and becomes a coupling factor for oxidation and PPi synthesis. It seems that phospholipid is indispensible for incorporation into the membrane and the manifestation of the coupling activity of the enzyme. The effect of lipids on the activity of soluble and membrane-bound pyrophosphatase was studied. It is shown that PPase II phospholipid is involved in the regulation of the hydrolase activity of the isolated enzyme. However, hydrolysis of PPi by SMP and its synthesis by mitochondria are affected by cooperative rearrangements of the entire lipid component of the membrane rather than by changes in the phase state of phosphatidyl choline contained in PPase II. An opposite response of ATP and PPi synthesis to changes in viscosity makes it likely that the viscosity of the mitochondrial inner membrane may control the levelling of these two processes in mitochondria.

The content of acid-soluble nucleotides was different in the cells of non-sulphur purple bacterium Rhodospirillum rubrum, growing phototrophically under anaerobic conditions and aerobically under conditions of heterotrophic nutrition. The cells of the culture growing in the darkness contained more adenylic and guanylic nucleotides, and the total of reduced and oxidized NADP. The cells of the culture growing in the light contained more FMN. The cells of the "light" and "dark" cultures contained GTP dependent phosphopyruvate carboxylase (E.C. 4.1.1.32). The cells of the "light" culture, contrary to the "dark" culture, contained also pyrophosphate dependent phosphopyruvate carboxylase (E.C. 4.1.1.38).

The biosynthesis of polyphosphatase and the system of active transport of glucose is repressed in the cells of Neurospora crassa by glucose at a high concentration. There is a strict correlation between the activity of polyphosphatase and the initial rate of glucose active transport. Both systems are repressed during the growth of the mycelium on a glucose medium and are repaired on a medium without glucose. The latter process is inhibited by cycloheximide. Under various conditions of cultivation, the ratio between the activity of polyphosphatase and the initial rate of active transport of glucose remains close to unity. The experiments with 8-azaadenine have shown that the system of active transport of glucose does not require ATP. A possible physiological role of polyphosphatase in N. crassa is discussed.

In yeast Saccharomyces N.C.Y.C. 644SU3 the simplest energy donor, inorganic pyrophosphate, can be formed not only in the processes coupled with the respiratory chain, but also under conditions when mitochondrial synthesis of the compound is inhibited (glucose repression, anaerobiosis). Stimulation of inorganic pyrophosphate formed after exogeneous addition of glycolytic system components and inhibition studies suggest that under those conditions the non-mitochondrial energy-dependent formation of inorganic pyrophosphate is due to glycolytic phosphorilation.

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.

The effect of respiration rate on the content and accumulation of ATP and PPi in rat liver mitochondria was studied. It was found that the syntheses of ATP and PPi occur at different respiration rates. It was assumed that the respiration rate can determined the formation of ATP or PPi via oxidative phosphorylation. A possible mechanism of this effect is discussed.

The chromatophores of Rhodospirillum rubrum were found to synthesize in the light not only ATP and pyrophosphate but also high molecular weight polyphosphates. Biosynthesis of all studied compounds was inhibited by antimycin A, an inhibitor of the electron-transport photosynthetic chain. Synthesis of high molecular weight polyphosphates is stimulated, while that of pyrophosphate is inhibited, in the conditions providing intensive synthesis of ATP (in the presence of ADP and in the absence of oligomycin). The results obtained suggest that biosynthesis of high molecular weight polyphosphates is related to photosynthetic phosphorylation, via ATP but not pyrophosphate.