[Analysis of free-radical products of near-UV irradiation at 77 K of frozen adenine and adenosine diphosphate solutions containing inorganic phosphate].

Irradiation by near-UV light at 77 K of aqueous solutions of inorganic phosphate in weakly acidic conditions in the presence of the photosensitizers adenine and adenosine diphosphate results in the formation of free radicals of these compounds, photosensitized free radicals of phosphate itself, and H* and OH* radicals. The relative concentrations of free radical products were estimated by the analysis of total ESR signals registered in the region of g = 2.00 in the photosystems Ade + Pi and Adphi + Pi using the original computer program of ESR spectra simulation.

[Energizing of F1-ATPase after irradiation with visible light]. / Energizatsiia F1-ATFazy pri obluchenii vidimym svetom.

It was shown that ATP synthesis by F1-ATPase sensitized by visible light was combined with oxidation of SH groups and decrease of initial flavin fluorescence in the F1-ATPase preparation. It was suggested that it was an endogenous flavin group that regulated redox transitions between SH-S-S groups which was essential for the catalysis in vivo.

[Electron paramagnetic resonance of the F1-H+-ATPase complex and adenosine triphosphate synthesis after its irradiation with visible light]. / Elektronnyi paramagnitnyi rezonans kompleksa F1-H+-ATPazy i sintez adenozintrifosfata pri ego osveshchenii vidimym svetom.

Under F1-ATPase irradiation by visible light two characteristic features of flavin main activities were detected: I/ESR signal g = 2.00 appearance which was in favor of flavin photochemical electron reduction and 2/photostimulated O2 consumption by F1-ATPase. The dependence of ESR signal g = 2.00 intensity on visible light wavelength completely coincided with the same dependence obtained for proteinless model riboflavin + ADP. Flavin localization in proximity of ADP bound in the enzyme active site was suggested. Under F1-ATPase irradiation by light lambda greater than 350 nm ATP synthesis was obtained similar to the proteinless model riboflavin + ADP + Pinorg. In this model endogenous flavin was suggested to serve as a photosensitizer, its photoexcitement being a model of dark energization of F1-ATPase in oxidative phosphorylation.

[Redox regulation in ATP synthesis]. / Okislitel'no-vosstanovitel'naia reguliatsiia v sinteze ATF.

Evidence is considered which points to changes of redox potential of the redox-centres in mitochondria during energization, to high sensitivity of ATP-synthetase to redox agents. Examples of ATP-syntheses in model systems stimulated with an electron are discussed. This stimulation is so efficient that it permits weakening of the bond between phosphorus atom and extremely bad leaving group O- in inorganic phosphate-phosphorylating agent in ATP synthesis during oxidative phosphorylation. The sum of these data suggests that function of the redox-centres found in the coupling site may be the accumulation of the intermediate inducing ADP and Phinorg interactions. The electron pool may serve as an intermediate. Thus the redoxcentre function in the coupling site accepting and accumulating the electrons during energization may be compared with chlorophyll function in photosynthesis. Change of redox potential of redox-centres at energization (by protonation, for example) initiates electron transfer in ATP-synthetase, which by the formation of highly reactive-free radical of ADP provides the occurrence of endergonic reaction of ATP synthesis, i.e. storage of energy as a chemical bond.

[Effect of visible light on the ATP synthetase function of mitochondria]. / Vliianie vidimogo sveta na ATF-sintetaznuiu funktsiiu mitokhondrii.

Dependence of oxidative phosphorylation in rat liver mitochondria on redox state of endogenous flavoenzymes was studied. Regulation of the redox state of endogenous flavins in mitochondria was performed by visible light illumination of lambda = 380 nm in oxygen free as well as in oxygenated media. A decrease of oxidative phosphorylation was obtained at full reduction of flavins (respiratory control and ATP synthesis were measured); this effect was partially recovered when flavin was reoxidized by oxygen. Results of ESR measurements of the signal gl = 2.00 associated with semireduced flavins were in accordance with the appearance of diamagnetically completely reduced form of flavin. These facts may be considered as evidence of the regulative role of flavins in oxidative phosphorylation.

[Electron spin resonance of phosphorylating and non-phosphorylating submitochondrial particles]. / Elektronnyi paramagnitnyi rezonans fosforiliruiushchikh i nefosforiliruiushchikh submitokhondrial'nykh chastits.

Paramagnetic centres related with the work of ATP-synthetase, found earlier on mitochondria were studied on phosphorylating and non-phosphorylating submitochondrial particles (SMP). A complex doublet signal with half-width 38 . 10(-4) T was shown to be recorded only on actively phosphorylating and oligomycin-inhibited SMP. It was found that the signal components were conditioned by different paramagnetic centres, one of which (g = 2.03) seemed to be the metal centre, more probable non-hemeiron. The nature of the second signal characterized by evenly saturating doublet components with g = 2.03 and g = 2.00 is unclear. Studies of flavin signal saturation have also shown that it consists of two components: one of them--saturating, specific for non-phosphorylating SMP, another--non-saturating, mostly observed in phosphorylating SMP, probably conditioned by flavin--the constituent of ATP-synthetase.

[Adenosine triphosphate synthesis induced by ultraviolet irradiation]. / Sintez adenozintrifosfata, stimulirovannyi ul'trafioletovym oblucheniem.

Effect of UV-irradiation on non-aqueous solutions of adenine nucleotide mixture of the presence of inorganic phosphate was studied. Solutions of ADP sodium salts containing AMP and ATP admixtures and of inorganic phosphate in DMSO and DMPA and ethylene glycol were illuminated with the lamp dPIII-500. It has been shown that along with nucleotide disintegration and complex formation ATP synthesis takes place, which is evidenced by an increase of relative ATP content in experimental specimens.

[Detection of a paramagnetic product developing during oxidative phosphorylation in mitochondria]. / Obnaruzhenie paramagnitnogo produkta, voznikaiushchego pri okislitel'nom fosforilirovanii v mitokhondriiakh.

It was shown by ESR technique using flow system combined with ESR-spectrometer that paramagnetic product appearing in the course of oxidative phosphorylation was directly associated with mitochondrial ATPase operation. A decrease of ESR signal intensity and the changes of its form observed on mitochondria uncoupled by 2,4-dinitrophenol as compared with those inhibited by olygomicin suggest that in the case of olygomycin block a free radical ATPase linked intermediate can be recorded, the ESR signal of which seems to be partly due to flavin semiquinone of ATPsynthetase itself.

[ESR study of mitochondria in basal metabolic states and at different levels of ATP-synthetase activity]. / Issledovanie mitokhondrii v osnovnykh metabolicheskikh sostoianiiakh pri razlichnykh urovniakh aktivnosti ATF-sintetazy metodom EPR.

Effect of metabolic transitions of mitochondria on the value of ESR-absorption measured at room temperature has been studied. It has been found that the content of free radicals with ESR signal of g = 2.00 and the halfwidth about 15e depends on the metabolic state of mitochondria determined by the load of respiration and phosphorylation systems of mitochondria. The maximal gain of the free radical content is observed in the active state when the maximal load is applied to the phosphorylative chain. An attempt to localize the region of initiation of additional free radicals by means of uncouplers and inhibitors of phosphorylation, as well as by placing mitochondria into the phosphate-free medium has shown that the change in ESR absorption of mitochondrion preparation is in a specific correlation with the change in energization of mitochondria. The analysis of alternative ways for an increase of the free radical fund during energization makes it possible to connect this increase with the work of ATP-syntetase and to suggest that free radicals take part in the ADP phosphorylation by inorganic phosphate, being probably, localized directly on the ADP molecule.

[EPR study of oxidative phosphorylation in surviving mitochondria]. / Issledovanie okislitel'nogo fosforilirovaniia v perezhivaiushchikh mitokhondriiakh metodom EPR.

Rat liver mitochondria were studied by ESR method in an original flow-type system, which allowed to maintain adequate conditions for survival. Low sensitivity of modern ESR-spectrometers for humid samples requires high concentrations of mitochondrial protein, which puts additional requirements for the survival of the sample. It is most important to create the level of oxidation substrates, phosphate acceptors and oxygen content sufficient for the normal course of oxidation and phosphorylation. The flow-type system applied makes it possible to overcome the above difficulties and permits biochemical control over the object state to be realized throughout the ESR spectrum recordings. Estimation of oxidative and phosphorylating activity of mitochondria has shown that the system makes it possible to realize all the metabolic transitions up to their maximal stimulation when passing to an active state.

[Participation of free radicals in ATP synthesis]. / Uchastie svobodnykh radikalov v sinteze ATF.

To study ESR absorption of mitochondrial ATPase a special flow system was developed allowing to maintain surviving conditions for mitochondria. It has been shown that ESR free radical signal of mitochondria observed at room temperature (g-factor 2.00 and halfwidth of about 15 Gs) depends on their metabolic state. An increase of free radical content during mitochondrial energization can be associated with operation of ATPsynthetase. It is supposed that free radicals take part in the reaction of ADP phosphorylation and that a molecule of ADP itself bound in the active centre of ATPase can become a free radical to facilitate the process of inorganic phosphate incorporation.