[On specific properties of the mitochondrial oxidative phosphorylation system operating as a supercomplex].

This paper represents the study of endogenous and exogenous fatty acids affecting the mitochondrial phosphorylation system effectiveness depending on temperature. The experiment was set up under conditions in which the oxidative phosphorylation system operates as a supercomplex. Rat liver mitochondria were isolated without purposive fatty acids removal from membranes, then studied in hypotonic medium (120 mOsm). We managed to detect a very narrow interval 19 ± 1°C where the fatty acid uncoupling effect is weak up to disappearing. At the same small temperature range, a structural rearrangement that takes place in the enzyme system is accompanied with denser packing of membrane protein complexes. Thus, at the temperatures close to 19°C the supercomplex works in the specific regime protected (or partially protected) from the uncoupling effect of fatty acids. Here we also discuss a physiological significance of the increased ATP-synthesis effectiveness at lower temperatures and the most probable character of structural rearrangement taking place at 19°C in the enzymes in the mitochondrial membrane.

[Interaction of surface-active base with fraction of membrane-bound Williams's protons].

In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

[Generation of reactive oxygen species in water under exposure of visible or infrared irradiation at absorption band of molecular oxygen].

It is found that in bidistilled water saturated with oxygen hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sourses, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.

[Detection of new two-membrane structures in native mitochondria by the method of small-angle scattering of neutrons].

The structure of mitochondrial cristas has been studied for the first time by the method of small-angle scattering of thermal neutrons. Experiments were performed on intact functioning rat liver mitochondria. Mitochondrial cristas are usually considered as folds of the internal membrane with arbitrary nonfixed intermembrane distances. It was shown that, under particular conditions, during low-amplitude swelling of mitochondria, cristas are transformed to bimembrane structures, with the distance between the central planes of membranes of 190 E. It was found that the formation of bimembrane structures and their structural parameters do not depend on the method of induction of swelling, by placing the mitochondria into a hypotonic medium or by the opening of nonspecific pores.

[Dynamics of mitochondrial ultrastructure in small pieces of cardiac tissue under long-term anoxic incubation].

Anoxic incubation of isolated small pieces of cardiac tissue for 72 h caused emergence of an unusual population of mitochondria, referred to as "mitochondrion inside mitochondrion". We studied dynamics of the origin of this event. In the most part of a mitochondrial population after a 6 h anoxic incubation of myocardial tissue, a local increase in some region of the intermembrane space was observed. Some regions of matrix with adjoined inner membrane move into these regions of intermembrane space, to be constricted eventually. After 12 h of incubation densely neighbouring layers of membrane are observed in these structures. By 24 h of incubation, inside new-formed structures well-distinguished concentric layers of membrane appear. Between these layers some electron-dense material ultrastructurally identical to mitochondrial matrix is seen. By 72 h of anoxic incubation, in cardiomyocytes of the experimental tissue structures with well-marked morphological features of mitochondria appear, which we called "mitochondrion inside mitochondrion". Results of our study are discussed in terms of a conception of changes that occur in the structure of mitochondrial reticulum during apoptosis.

[IR-luminescence of low-stability aqueous structures (theory and experiment)]. / IK-liuminestsentsiia vodnykh struktur s nizkoi ustoichivost'iu (teoriia, éksperiment).

The effect of IR luminescence in liquid water was registered. A theory was suggested according to which the bands of IR luminescence are related to the presence of clusters (clathrates) in water. The effect arises in water exposed to low-intensity electromagnetic oscillations. Under strong exposure, the decomposition of luminescent structures takes place. A quantitative theory was proposed and the spectrum of water luminescence for the region of strain oscillations was calculated. A good agrement between the theory and experimental data was obtained.

[Local coupling of respiration processes and phosphorylation in rat liver mitochondria]. / O lokal'nom sopriazhenii protsessov dykhaniia i fosforilirovaniia v mitokhondriiakh pecheni krysy.

One variant of the model of the local coupling of phosphorylation and respiration in intact mitochondria was experimentally verified. The model is based on the following postulates: (1). Upon the functioning of H+ pumps, hydrogen ions bound to the outer membrane surface do not enter the aqueous phase but are utilized for ATP synthesis in the membrane supercomplex respiratory H+ pump--ATP synthetase. (2). During the functioning of H+ pumps, an appreciable part of the energy of oxidation reactions can be stored in the form of the thermodynamic (solvation) potential of H+ ions bound to the outer membrane surface. According to the model, the hydration of hydrogen ions during the transition from the outer face of the inner membrane to the aqueous phase should lead to a decrease in the efficiency of the system of the coupling of respiration and phosphorylation. The model takes into account the ability of the nonpermeating buffer to catalyze the detachment of hydrogen ions from the membrane surface to the aqueous phase and provide their complete solvation. A preparation of phosphorylating mitochondria with the covalently bound pH probe was obtained. This made it possible to register for the first time the presence of a local H+ gradient on the outer side of the inner mitochondrial membrane during the stable functioning of the oxidative phosphorylation system. It was shown on these mitochondrial preparations that a decrease in the outer local H+ gradient by the action of increased concentrations of buffer is accompanied by a significant decrease in the ADP/O parameter and a partial dissociation of oxidative phosphorylation. Conditions were determined under which increased concentrations of buffer in the incubation medium cause a partial dissociation and a decrease in the ADP/O value from 20% to twofold (depending on the quality of mitochondrial preparations). The results obtained are in full agreement with the predictions of the model.

[Effect of superlow quantities of hydrogen peroxide on water base of solutions]. / Vliianie sverkhmalykh kolichestv peroksida vodoroda na vodnuiu osnovu rastvorov.

In was shown previously that cations in superlow concentrations strongly change the integral characteristics of the state of the solution water component. The role of the structure of the water component in this process was examined using a model of hydrogen peroxide transformation in water. The dispersion rates of the transmission factors for nine regions of the IR spectrum were analyzed in hydrogen peroxide solutions of different superlow concentrations having the Makhalanobis criteria maximal and close to the standard, which indicate the integral state of the water component in the presence of this substance.

[Detection and functional role of local gradients of H+-ions on the intracellular mitochondrial membrane with covalently linked pH-probe]. / Obnaruzhenie i funktsional'naia rol' lokal'nykh gradientov H+-ionov na vnutrennei membrane mitokhondrii s kovalentno prisoedinennym H-zondom.

The study is devoted to the registration of local H+ gradients on the inner membrane of mitochondria under conditions of H+ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K+ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H+ gradients on the mitochondrial membrane. It was concluded that local gradients of H+ activity can control the rate of functioning of H+ pumps. It was shown that, under certain conditions, the system of H+ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system.

[Fluctuations of pass band coefficients of water and water salt solutions in the spectral infrared region]. / Fluktuatsii koéffitsientov propuskaniia vody i vodnykh rastvorov solei v infrakrasnoi oblasti spektra.

The effect of superlow concentrations of KCl and CaCl2 solutions, obtained by diluting starting 1 M solutions of these salts 10(9)-10(15) times, on the fluctuations of pass band coefficients of water was studied. It was found that these solutions are substantially distinguished from bidistilled water by a high intensity of fluctuations of spectra in the infrared region. It was also shown that higher concentrations of solutions of these salts (at dilutions less than 10(8) times) have no strong and specific effect on fluctuations of the infrared spectrum: the intensity of fluctuations of the infrared spectra of these solutions practically coincides with that of control water. Possible reasons for this effect are discussed.

[Ultrastructure of mitochondria apparatus of cardiomyocytes in apoptosis induced by long-term anoxia in rats]. / Ul'trastruktura mitokhondrial'nogo apparata kardiomiotsitov krys pri apoptoze, indutsirovannym dlitel'nym deistviem anoksii.

Apoptosis in cardiomyocytes was induced by incubation of pieces of cardiac tissues under condition of anoxia. Electronmicroscopic investigation detected previously unknown changes in mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In addition to a mitochondrial population characterized with irrigated cleared matrix, anoxia induced the appearance of an atypical and previously unknown population of small electron-dense cardiomyocyte mitochondria. They were characterized by unusual localization inside electron-light mitochondria ("mitochondria inside mitochondria"). The most part of mitochondria with the irrigated matrix are commonly characterized by unusual types of rearrangements of the inner mitochondrial membrane. Under anoxic conditions, the inner mitochondrial membrane formed electron-dense ordered structures. This is a spongy structure with cells of equal size. Results of our study are discussed in terms of conception of changes in mitochondrial reticulum ultrastructure during apoptosis.

[Cooperative processes in mitochondria: registration by dyanmic phase microscopy]. / Kooperativnye protsessy v mitokhondriiakh: registratsiia metodom dinamicheski fazovoi mikroskopii.

The spectra of phase fluctuations in mitochondria were measured by the method of dynamic phase microscopy. Contrasting components were revealed whose intensity markedly changed at distances of 100-300 nm. Similar frequency components were observed in the spectra of ATP-stimulated fluctuations in liposomes with the incorporated ATPase. The values of the frequency and intensity of contrasting components in spectra of liposomes, mitochondria, and cells are presented. The possibility of determining the position of active enzyme complexes from their characteristic frequencies is discussed.

[Mitochondrial proteolipids]. / Proteolipidy mitokhondrii.

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 effect of inhibitors of the Q-cycle on cyano-resistant oxidation of malate by rat liver mitochondria in the presence of menadione]. / Vliianie ingibitorov Q-tsikla na tsianrezistentnoe okislenie malata mitokhondriiami pecheni krys v prisutstvii menadiona.

Based on the inhibitor analysis data, it has been assumed that the Q-cycle plays a role in the cyano-resistant malate oxidation induced by menadione (90 microM) in rat liver mitochondria. The extent of involvement of Q-cycle transmitters in the cyano-resistant respiration of mitochondria is determined by the mode of the electron supply into the Q-cycle. In the presence of dicumarol, i.e., under conditions when CoQ and menadione are reduced by NADH-quinone reductase, the bulk of the electrons pass through the o-center of the Q-cycle. Myxothiazole inhibits the respiration by 70-80%, while antimycin--by only 20-30%. In the presence of myxothiazole and antimycin menadione oxidizes cytochrome b. In the presence of rotenone, when menadione is reduced by DT-diaphorase, the rate of cyano-resistant respiration decreases approximately twofold; its sensitivity towards myxothiazole and antimycin drops down to 40%. In the absence of rotenone and dicumarol the Q-cycle does not participate in the cyano-resistant respiration which under these conditions is insensitive either to myxothiazole or to antimycin. It is concluded that the mechanism of cyano-resistant respiration changes with an alteration in the rates of quinones K3 and CoQ reduction. The mechanism of cyano-resistant respiration is also controlled by the medium tonicity. A reduction in the medium tonicity decrease the participation of the Q-cycle and, correspondingly, the sensitivity of the cyano-resistant respiration towards myxothiazole and antimycin.

[Five classes of anticarcinogens--phorbol ester antagonists]. / Piat' klassov antiopukholepromotorov--antagonistov forbolovykh éfirov.

Literary data concerning structure-activity relationships in compounds exhibiting an antipromoter activity in skin tests of full tumour promotion by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or its analogs have been reviewed. Five classes of antipromoters differing in their action on the first and second stages of tumour promotion have been identified, namely: i) 2nd stage promoters/1st stage inhibitors; ii) 1st stage promoters/2nd stage inhibitors; iii) 2nd stage inhibitors; iiii) 1st stage inhibitors; iiiii) 1st and 2nd stage inhibitors. The results on structure-activity of antipromoters were compared with the results of previous studies on phorbol tumour promoters. These data made it possible to determine the reciprocal localization of binding sites for various classes of antipromoters and individual functionally important phorbol fragments on protein kinase C. A new detailed variant of the model of two-center binding of full phorbol tumour promoters on protein kinase C is proposed.

[The effect of the composition of phospholipid bilayer membranes on the steady state kinetic transfer of protons]. / Vliianie sostava bisloinoi fosfolipidnoi membrany na statsionarnuiu kinetiku perenosa protona.

When the hydrogen ion flux was induced by nigericin across the planar bilayer lipid membrane (BLM) with the bulk pHs being equal at the opposite sides of the BLM, the formation of the boundary potential difference (delta phi b) on the BLM was observed using the method of the inner membrane field compensation. The increase in the buffer (citrate, phosphate, MES) concentration caused a decrease in the delta phi b. The latter is generated in the presence of phosphatidylserine in membrane-forming solutions only. It was assumed that the steady state difference of the surface hydrogen ion concentration is created under conditions of equal pHs near the BLM surface. In another experimental series the monensin-mediated formation of the surface protein concentration difference gave rise to the formation of a potassium ion gradient induced by nigericin. Thus, a possibility of performing work due to the surface proton concentration gradient was demonstrated. The relevance of these findings to the processes of energy transduction during membrane phosphorylation in mitochondria and chloroplasts is discussed.

[Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]. / Vzaimodeistvie menadiona i durokhinona s Q-tsiklom v usloviiakh rabotyDT-diaforazy

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.

[Changes in the level of tightly-bound mitochondrial phospholipids induced by exogenous factors]. / Izmenenie soderzhaniia prochnosviazannykh fosfolipidov mitokhondrii pod vliianiem ékzogennykh faktorov.

The effects of some parameters of the incubation medium (tonicity, H+ and Pi concentration) on the phospholipid content in rat mitochondrial extracts prepared by normal extraction and by extraction with strongly acidified solvent mixtures, were studied. It was found that after normal extraction with a chloroform-methanol mixture, 8% to 25% of mitochondrial phospholipids can be additionally extracted by acidified solvent mixtures. The concentration of weakly extractable lipids depends on the tonicity and ionic composition of the incubation medium. It was suggested that there exist in mitochondria two lipid pools, a loosely and a tightly-bound ones; the equilibrium between these pools is effectively regulated by medium tonicity and other external factors.

[Complexes of phorbol tumor promoters with protein kinase C]. / Kompleksy opukholepromotorov forbolovogo riada s proteinkinazoi C.

Based on the literature data, a systematic comparison of relationships between the structures of incomplete and complete phorbol tumor promoters, diacylglycerols and their activities in various biological test-systems was carried out. The specific features of the phorbol esters-protein kinase C complexes responsible for the induction of the first and second stages of the tumor promotion in mouse skin, were established. The type of diacylglycerol binding to protein kinase C which confers to the latter noncarcinogenic properties, was specified.

[Two qualitatively different structuro-functional states of mitochondria]. / Dva kachestvenno razlichnykh strukturno-funktsional'nykh sostoiania mitokhondrii.

Low (120 mosM) tonicity of incubation media of mitochondria was found to be associated with anomalous phase transition at 19--26 degrees C. A rise in temperature caused a decrease in the pyrene excitation in border lipids of the mitochondrial membrane. Within this temperature range the quenching of intrinsic protein fluorescence by pyrene was sharply decreased. It may be inferred from these data that at 100mosM tonicity and temperatures below 19 degrees C, mitochondrial membrane proteins are in an aggregated state. At temperatures above phase transition protein deaggregation takes place. It was shown that a decrease in tonicity from 300 to 120 mosM at 15 degrees C or a rise in temperature from 15 degrees to 37 degrees C at 300 mosM tonicity increased the phosphorylation of the 52 kDa mitochondrial protein. It was assumed that swelling of mitochondria in hypotonic media simulates one of the steps of the hormone-induced signal transfer in mitochondria in vivo.