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Alkaline version of the single-cell gel electrophoresis (Comet assay) is widely used in toxicological, environmental, and monitoring studies to assess the DNA damage levels in individual cells. The change in the temperature of the electrophoretic solution is one of the reasons leading to interlaboratory variation of Comet assay results. In this work, changes of surface temperature of the solution during electrophoresis were studied using technique of real-time thermal imaging. It has been found that the electrophoresis is accompanied by nonuniform temperature rise in different areas of the electrophoretic chamber. The maximum of heating was observed in the central region of the chamber, where temperature increased by an average of 7°C. The minimum temperature rise in other parts of the chamber was about 5°C. After removing the solution, the temperature on the surface of slides was higher than that on the surface of the solution. We believe that (1) nonuniform heating of the electrophoretic solution and convection could be the reasons responsible for the variability of results both in inter- and intralaboratory studies; (2) the spatial distribution of heating of the solution depends on the size and configuration of the electrophoretic chambers used., (© 2021 Wiley-VCH GmbH.)

Purpose: The objective of the study was to estimate the DNA damage in blood leukocytes at long terms after irradiation of mice with carbon ions (450 MeV/nucleon) both before and in the Bragg peak., Materials and Methods: White outbred SHK male mice were exposed to whole-body irradiation with carbon ions at doses of 0.1-2 Gy in the spread-out Bragg peak and at a dose of 6 Gy before and in the Bragg peak. At different times after irradiation (1-75 days), whole blood was collected from the tail of each mouse and analyzed by the comet assay. Mice X-irradiated in the same dose range served as a positive control. The level of the expression of mRNA of CDKN1A, APEX1, BBC3, TXN2, and β-ACT genes in bone marrow cells was determined in animals irradiated with carbon ions at doses of 0.1-2 Gy using the real-time PCR., Results: It was found that, 24 h after 12 C-irradiation, a dose-dependent (0.1-2 Gy) increase in the DNA damage of leukocytes occurred, which was accompanied by a decrease in their concentration and an increase in the expression of the CDKN1A and BBC3 genes in bone marrow cells. The expression of the APEX1 and TXN2 genes did not change. In mice 12 C-irradiated at a dose of 6 Gy before and in the Bragg peak, the level of DNA damage changed as follows: by day 3, it increased; by day 23 it returned to the control level; by day 30, it increased again; and by day 75, it fell to the control level on irradiation before the Bragg peak and was significantly higher ( p < .05) than in the control after irradiation in the Bragg peak., Conclusions: The dynamics of changes in the level of DNA damage in leucocytes of 12 C-irradiated mice within 30 days is similar to that in mice exposed to sublethal doses of X-radiation. The retention of the high level of DNA damage by day 75 after 12 C-irradiation in the Bragg peak indicates a significant injury of cells from different cell pools of the blood system. The high level of DNA damage may be related not only to complex DNA injuries but also to chronic oxidative stress.

The superoxide-generating reaction of adrenaline autoxidation in an alkaline medium, used in vitro to identify the antioxidant properties of various compounds, simulates the complex multistep process of quinoid oxidation of catecholamines (CA) in the body. Sulfur-containing cysteine (Cys) and reduced glutathione (GSH), as well as oxidized glutathione (GSSG), have been shown to inhibit this process. The studied substances were considered as inhibitors of quinoid oxidation and are evaluated as antioxidants. The IC50 values for Cys and GSH were close to 7.5 mM. Inhibition by GSSG was weaker; represented approximately 50-70% of Cys and GSH. Other sulfur-containing compounds that differ in chemical structure, the amino acids taurine and methionine were ineffective. The interest in this model and the search for effective compounds acting on this reaction is associated with one of the mechanisms of the etiopathogenesis of Parkinson's disease (PD) discussed in the literature, which occurs when the biochemical transformations of dopamine CA and its quinoid oxidation process are violated. Cys, GSH and GSSG in the model system inhibit quinoid oxidation of adrenaline, as a result of which the formation of superoxide (O2 ·-) is also inhibited. Experiments with the superoxide-generating enzymatic reaction xanthine xanthioxidase, the chemistry of which is different and not related to formation of quinoid metabolites, showed that the studied substances did not inhibit O2 ·- formation in this model. Thus, it was established that the biologically active sulfur-containing compounds Cys, GSH and GSSG are specific inhibitors of quinoid oxidation of CA, and are likely to be able to play the role of a neuroprotector. It is proposed to use these compounds in the treatment and prevention of PD by activating their biosynthesis in the body.

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of the activity of superoxide dismutase and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first is based on registration of adrenochrome, as adrenaline autooxidation product at 347 nm; the second employs nitro blue tetrazolium (NBT) and registration of diformazan, a product of NBT reduction at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been proposed. The main approach consists in the use of the pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some features in kinetic behavior of its autooxidation; they are applicable in the superoxide generating system based on adrenaline autooxidation. Performing measurements at 560 nm, the reaction rate can be regulated by lowering the concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this cannot be done. These features of adrenaline autoxidation may be due to the fact that the intrinsic multistage process of the conversion of adrenaline to adrenochrome, which is recorded at 347 nm, is coupled with the transition of electrons from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions, which is detected in the presence of added NBT.

An important role of carbonate/bicarbonate ions has been recognized in the superoxide generating reaction of adrenaline autooxidation in an alkaline buffer (a model of quinoid adrenaline oxidation in the body). It is suggested that these ions are directly involved not only in formation of superoxide anion radical (О(2)(-)) but also other radicals derived from the carbonate/bicarbonate buffer. Using various buffers it was shown that the rate of accumulation of adrenochrome, the end product of adrenaline oxidation, and the rate of О(2)(-)· formation depend on concentration of carbonate/bicarbonate ions in the buffer and that these ions significantly accelerate adrenaline autooxidation thus demonstrating prooxidant properties. The detectable amount of diformazan, the product of nitro blue tetrazolium (NBT) reduction, was significantly higher than the amount of adrenochrome formed; taking into consideration the literature data on О(2)(-)· detection by NBT it is suggested that adrenaline autooxidation is accompanied by one-electron reduction not only of oxygen dissolved in the buffer and responsible for superoxide formation but possible carbon dioxide also dissolved in the buffer as well as carbonate/bicarbonate buffer components leading to formation of corresponding radicals. The plots of the dependence of the inhibition of adrenochrome and diformazan formation on the superoxide dismutase concentration have shown that not only superoxide radicals are formed during adrenaline autooxidation. Since carbonate/bicarbonate ions are known to be universally present in the living nature, their involvement in free radical processes proceeding in the organism is discussed.

In this paper the oil from seeds of Amaranthus cruentus L. (AmO) was shown to be an efficient modulator of the physical chemical properties of artificial lipid and rat hepatocyte plasma membranes. AmO improved the membrane stability, their stress resistance and the adsorption of neurotensin to plasma membranes with the distinct biphasic interactions being observed even after adrenalin stress exposure. The analysis of pro-/antioxidant balance in rat blood revealed a mild prooxidant activity after AmO intake, which was accompanied by accumulation of oxidative destruction products in plasma membranes. This prooxidant action of AmO was corroborated in vitro in an adrenalin autooxidation model. On the other hand, the observed improved resistance to adrenalin stress in AmO supplemented rats was associated with an antioxidant response in blood and plasma membrane studies. The AmO effects can be attributed to the modulation of the metabolic pathways involved into oxygen and free radical homeostasis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.

Addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows to identify directly superoxide anion formation (02.-) in this superoxide-generating system. The kinetics of formation of adrenochrome and 02.- were compared under the same conditions. Three possible approaches to the use the adrenaline autooxidation reaction for determining the activity of superoxide dismutase (SOD) and revealing the antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectrophotometric method of the registration of adrenochrome, the final product of adrenaline autoxidation, at 347 nm (Sirota, 1999) and the polarographic method, based on determination of oxygen consumption for 02.- formation (Sirota, 2011). Here, a novel approach to this problem is presented; it consists in the spectrophotometric determination of 02.- using NBT. The application of this approach enables one to lower the pH of carbonate buffer from 10.5 to 9.7 and to decrease (4-fold) the amount of added adrenaline, i.e., to generate milder and more effective conditions for revealing and studying the antioxidant properties of examined materials.

The reaction of adrenaline autoxidation in an alkaline buffer with the formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinoid pathway of adrenaline conversion in the body, is accompanied by oxygen consumption. This reaction is applicable for polarographic determination of the activity of superoxide dismutase and the antioxidant properties of biological and chemical compounds, it is based on evaluation of the latent period and the rate of oxygen consumption, which are measured in the presence of the compounds examined. It was assumed that the neuro- and cardiotoxicity of quinone products of adrenaline oxidation is related not only to their "own" properties and reactive oxygen species formed but also the hypoxia of those regions of the cell and tissue where the quinoid oxidation of adrenaline occurs.

The ability of water preparations of different composition to affect the generation of superoxide radicals has been compared. The superoxygenating reaction of adrenaline autoxidation with some modifications was used as a model system, which makes it possible to reveal the pro/antioxidant properties of materials being tested. It was shown that samples of water from sources having different specific electroconductivity and, accordingly, ionic composition differ in the ability to affect reactions proceeding with the participation of ROS. The parameter measured, the pro/antioxidant activity of water, is a new informative indicator, and the approach proposed enables one to perform a comparative estimation of the quality of water and aqueous solutions.

The antioxidant system of some tissues in guinea pigs with no selenium deficiency was studied after treatment with selenium-containing products. Activity of a selenium-containing enzyme glutathione peroxidase was significantly reduced in the kidneys and blood. The kidneys were characterized by low activity of glutathione reductase and high activity of SOD. These features illustrate the development of oxidative stress in the kidneys.

The effect of negatively charged ions on respiratory organs and blood of rats has been studied. It was shown that the inhaling of negative air ions (NAI) for 60 min with a concentration of NAI at the place of location of animals 320-350 000 ions/cm2 activated the secretion of goblet cells without damaging the mucosa of the trachea and changed the spectrum of proteins of bronchopulmonary lavage. It was also found that the spontaneous production of reactive oxygen species (ROS) by cells of nonfractionated blood after the exposure to NAI increased in both males and females; the intensity of ROS generation induced by opsonized zymosan increased only in females. Different sensitivity of the antioxidant enzymes superoxide dismutase and glutathione reductase of blood to NAI in females and males was revealed. These results enable one to consider the effect of NAI as priming and a weak activation of the respiratory organs through the direct action on the mucosa of the primary target organs of the respiratory tract and then on the blood.

We studied behavioral reactions of rats after injection of subconvulsive dose of kainic acid into the frontal cortex and mitochondrial respiration in the hippocampus and frontal and temporal cortex 17-20 days after administration of kainic acid. Retention of acquired habit and the dynamics of its extinction in experimental rats were close to those in the control group. Changes in mitochondrial function were observed only in the region of kainic acid injection: activation of phosphorylating respiration during oxidation of succinate. Presumably, the detected activation of energy metabolism in the frontal cortex indicates functional restructuring in mitochondria, aimed at compensation of disorders caused by the neurotoxin.

It has been shown that a three-week feeding of rats with oil derived from seeds of amaranth (Amaranthus cruentus L.) leads to a moderate activation of respiration of coupled and uncoupled rat liver mitochondria (MCh) that oxidize succinate and succinate + glutamate, as well as alpha-ketoglutarate and alpha-ketoglutarate + malonate. In animals receiving the amaranth oil, the injection of adrenaline did not affect the oil-activated respiration of MCh during succinate oxidation; i. e., animals prepared by an oil-enriched diet were resistant to the action of adrenaline, which prevented from possible hyperactivation of mitochondrial functions. In the group of control animals, which received no oil, the injection of adrenaline activated the rate of phosphorylating respiration of MCh during oxidation of succinate or succinate + glutamate: the rate of oxygen uptake in state 3 respiration (by Chance) increased, and the phosphorylation time decreased. The injection of adrenaline did not affect the parameters of respiration of MCh that oxidize a-ketoglutarate; however, in the presence of malonate, the oxidation of alpha-ketoglutarate in state 3 and uncoupled respiration have shown mild but significant increase in response to adrenaline. In animals receiving the amaranth oil, the oil-induced activation of respiration of MCh in response to adrenaline retained but did not increase; however, the phosphorylation time significantly decreased. Thus, concentrated oil of seeds activates the respiration of MCh. In addition, it enhances an energetic function of MCh, which prevents from the hyper-activation of mitochondrial respiration by adrenaline. Therefore an activation of energetic function of MCh by amaranth oil could explain its adaptogenic effect on rats.

Cognitive processes and functional state of mitochondria in brain structures of Wistar rats were studied after intrahippocampal injection of kainic acid, an agonist of glutamate receptors. A single administration of 0.25 microg kainic acid into the dorsal part of the left and right hippocampi affected task retrieval and decreased inhibition of unrewarded responses. The injection of 0.75 microg kainic acid induced recurrent seizures and completely disorganized animal behavior. The functional state of mitochondria, as an important marker of excitotoxicity, was studied after intrahippocampal injections of kainic acid in the same doses. Kainic acid at 0.25 microg proved to activate the oxidative phosphorylation in hippocampal mitochondria. A higher (epileptogenic) dose of kainic acid inhibited mitochondrial respiration in the frontal cortex, but had an insignificant effect on mitochondrial respiration in the hippocampus. The disturbed interaction between the hippocampal system and frontal cortex after kainic acid administration can be the main factor of the revealed cognitive dysfunctions.

A new approach for evaluation of the antioxidant activity of lacrimal fluid (LF) has been proposed. It is based on the method of the analysis of the antioxidant activity of blood and superoxide dismutase activity (Sirota T.V., 1999). In the present work, the method has been first used for examining the antioxidant properties of LF, which is estimated by the ability of LF to inhibit or activate the rate of adrenaline autooxidation in an alkaline medium. The method does not require expensive reagents and equipment; it is simple, accessible, and highly informative. LF of practically healthy people possesses a marked antioxidant activity. In people with symptoms of acute respiratory virus infections, complete loss of the antioxidant activity of LF and the appearance of prooxidant activity were observed. In people taking polyvitamins in prophylactic doses, a significant increase in the antioxidant activity of LF was observed. This method may be applicable in ophthalmology for the diagnosis, control of treatment efficiency, and prognosis of eye diseases.

The role of impairment of general oxidative and energy metabolism in pathogenesis of lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia (BPH) and their correction by (1-adrenoblocker alfuzosin was studied. One group of patients (N = 126) was examined by standard methods for determination of the severity of LUTS by IPSS and mean effective volume of urinary bladder (MEVUB). In the second group (N = 29) in addition to functional examinations, metabolic indicators in blood were measured: antioxidant activity (AOA) and succinate dehydrogenase activity (SDA). Severity of LUTS depends greatly on the MEVUB. It was the first to show a practically complete correlation between LUTS, AOA and SDA. Severity of LUTS exactly correlates with indicators of oxidative and energy metabolism. In patients with more heavy LUTS, lowest AOA and SDA values were found. In the course of effective treatment, both phenomena developed an improvement of clinical symptoms and a rise of biochemical parameters. Close correlation between functional and metabolic phenomena is evidence of an essential role of metabolic mechanisms in the pathogenesis of LUTS with BPH. This opens perspectives to use antioxidants and energy metabolism activators for correction of UB dysfunction in patients with BPH.

It was shown that plantlets of wheat (Triticum vulgare) are capable of generating negative aeroions during the electrization of soil by high-voltage impulses. Soil electrization was carried out either from the moment of planting of seeds or from the appearance of the first seedlings. The concentration of negative ions was measured in the air at a distance of 50 cm from plants. In both variants, similar growth-related changes in the concentration of negative ions were observed. The generation of negative ions began on day 6 after the planting of seeds and reached a concentration of 380 x 10(3) ion/cm3. During the next three days, this level remained unchanged. On day 10, the generation of negative aeroions increased abruptly; on days 10-14, it was twofold as high as on days 7-9. The level of generation of negative aeroions by plants stimulated from the moment of appearance of plantlets was 5-8% higher than by plants stimulated from the moment of planting. The intensity of generation of negative aeroions upon additional illumination and in full darkness remained unchanged.

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.

A convenient model for studying the mechanisms of biological self-organization is described by morphometric investigation of formation of mitochondrion associations in medium containing physiological concentration of potassium ions without nonpolar substances. Association formation was considerably better at 15-18 degrees C during isolation and storage than at 0 degree C. The existence of filamented mitochondria in homogenate was also shown by staining of succinate dehydrogenase. Formation of associations increased in medium pretreated with negative air ions carrying superoxide and is probably due to hydrogen peroxide. The effect of substances influencing the surface charge on association formation was studied.

The intensive rise of absorption maximum at 347 nm (wave number 28.8) was found during autoxidation of low concentrations of adrenaline (230 microM) in alkaline pH (10.65) under room temperature and in the absence of additional sources of superoxide. It was stated that the appearance of this product of adrenaline oxidation, which was not reported earlier, is considerably more rapid than formation of adrenochrome (absorbtion at 480 nm) and is inhibited by superoxide dismutase (SOD). It was shown that erythrocyte hemolysate and some studied antioxidants (ascorbate and cysteine) inhibit formation of this substance like SOD. The determination of the described substance can be used for measurement of both: activity of SOD or antioxidant activity of different biological materials. The possibility to use available not expensive chemicals (0.1% adrenaline solution from drug store, 0.2 M bicarbonate buffer) serves as an additional advantage of the proposed method.

The effect of negative air ions (NAI) inhalation by rats on energy reactions of mitochondria in homogenates of liver and brain was studied. The influence of NAI was investigated under activation of animals by administration of physiological dose of adrenaline. Adrenaline administration induced hyperactivation of the rate of phosphorylating oxidation of succinic acid in liver and brain mitochondria which was accompanied by oxalacetate inhibition of succinate dehydrogenase (SDH) as well as excessive Ca2+ accumulation in liver mitochondria. These changes connected with a decrease of coefficient of phosphorylation efficiency ADP/O and uncoupler stimulation of respiration evidenced decrease of energy control of respiration in mitochondria. NAI inhalation diminished the rate of hyperactivated respiration and abolished excessive Ca2+ accumulation. These changes together with ADP/O coefficient and DNP stimulation increase evidence improvement of energy control of respiration which provide more moderate function of the respiratory chain under activation by adrenaline. Animals were excited by adrenaline administration. This effect was abolished by NAI inhalation, animals relaxed, some fell asleep. The data evidence sensitivity of mitochondrial processes in internal organs to inhalation of NAI and show participation of mitochondria in realization of physiological effects of NAI.

The effect of negative air ions (NAI) was investigated on physiological processes of peroxidative oxidation in mitochondria preserving their native structure organization in associations in rat liver homogenate. Tchijevsky lustre generating superoxide was served as a source of NAI. The investigated objects were put into solutions pretreated with NAI flow. An increase of peroxide induced Ca2+ release from mitochondria loaded with the cation was found. Under prolonged action of NAI flow on homogenates kept in ice followed by incubation in media pretreated with NAI an elevation of products of peroxide lipid oxidation (POL) was found in case of their low initial level and decrease of POL products in case of their high initial level. The range of changes is considerably less than under pathogeneous rise of POL. The discovered mild activation of peroxidative oxidation within physiological range of POL concentrations is considered as a primary physico-chemical mechanism of beneficial biological action of NAI.

The effect of oxygen partial pressure and solvent on the kinetics of beta-carotene consumption and oxygen uptake in the course of beta-carotene oxidation has been studied. The reaction of free radical formation, i.e., the chain initiation reaction that occurs via interaction of beta-carotene with oxygen and chain branching reactions that occur via interaction of intermediate products with oxygen, are found to be the most sensitive to the effect of environmental factors. These reactions determine the dependence of the beta-carotene consumption and oxygen uptake rates on oxygen partial pressure. The reaction rates increase in polar solvents which causes the increase in the beta-carotene consumption and oxygen uptake rates. The rates of peroxyl radical destruction and/or isomerization increase in polar media, which leads to the formation of low molecular weight polar oxidation products.

The effect of negative air ions generated by a Tchijevsky lustre was studied in rat liver mitochondria in homogenate under conditions providing preservation of their native structural organization in the form of associations. Rapid formation of large associations from intermediate and small associations occurs after the homogenate was put into medium pretreated by negative air ions (NAI). Under the influence of a flow of NAI on mitochondria stored on ice for 2-3 h formation of giant associations is observed. Associations of mitochondria are not dissipated by stirring with a magnetic stirrer for several minutes. The influence of medium pretreated with NAI or flow of NAI on homogenate improves weakened respiratory energy control and restores stimulation of phosphorylating respiration by adrenalin administration. The improvement of phosphorylation in mitochondria by NAI is perhaps caused by more complete preservation of both their native structural organization and related physiological regulation of mitochondrial processes in isolated preparations.

The activity of erythrocyte cytosolic superoxide dismutase from rat, bovine, man and duck was considerably increased when measured after preparation or incubation in media pretreated with negative air ions (mostly superoxide) from electroeffluvial ion generator. 0.5-1.0 microM H2O2 was found in incubation medium after treatment with air ions. The stimulatory effect of air ions on superoxide dismutase activity was mimicked by addition of 0.5-6 microM H2O2. The primary physicochemical mechanism of beneficial biological action of negative air ions is suggested to be related to the stimulation of superoxide dismutase activity by micromolar concentrations of H2O2.

Mitochondria in the form of associations close to their state in the quiescent cell were obtained in rat liver homogenate under physiological for cytosol concentrations of potassium and calcium ions and small dilution of tissue. Associations in such homogenates are stable on storage in ice for several hours. Dissociation of associations is induced by dilution of the homogenate, addition of EDTA, or by administration of adrenaline to the animal. Respiration rate is increased and ADP/O ratio is decreased when associations dissociate. The observed increase in respiratory rate induced by adrenaline administration in vivo is decreased or abolished when associations are dissociated in the incubation medium induced by influences in vitro. This is due to hyperactivation of respiration which leads to inhibition of succinate oxidation by oxaloacetate. Under the conditions preserving the associations of mitochondria a close interaction of oxidative and transamination processes is observed as well as a broader range of metabolic states of mitochondria than in standard preparations which dissipate into single granules on isolation in sucrose, high dilution, and washing.

Polyclonal rabbit antibodies against a Ca(2+)-binding mitochondrial glycoprotein were found to inhibit the uniporter-mediated transport of Ca2+ in mitoplasts prepared from rat liver mitochondria. Spermine, a modulator of the uniporter, decreased the inhibition. This glycoprotein of M(r) 40,000, isolated from beef heart mitochondria and earlier shown to form Ca(2+)-conducting channels in black-lipid membranes, thus is a good candidate for being a component of the uniporter. Antibody-IgG was found to specifically bind to mitochondria in human fibroblasts.

Single administration of Ca2(+)-transporting-Ca2(+)-binding glycoprotein simultaneously with intraperitoneal inoculation of Ehrlich ascites tumor cells into mice led to a decrease in the animals weight growth by approximately 34% as compared with the animals which were not treated with the glycoprotein. The effect observed was clearly pronounced, especially within the first days after inoculation of Ehrlich ascites tumor. Lifetime of individual experimental animals was increased whereas that of the whole group remained unchanged.

A rapid and sensitive method for protein determination (0.5-16 micrograms) in samples of any volume containing various surfactants in concentration up to 1% is suggested. The method includes the protein acid denaturation, the solution of acid insoluble precipitate of detergent in ethanol (25-30%), the protein determination on nitrocellulose filter, dyeing by aminoblack 10 B, elution of dyed complex and colorimetric determination at 630 nm.

The 40,000-dalton glycoprotein and 2000-dalton peptide inducing selective Ca2+-transport through bilayer lipid membranes were isolated from beef heart homogenate and mitochondria. Micromolar concentrations of these substances were found to increase the conductivity of membranes by 3-4 orders. Trans-membrane Ca2+ gradient induces an electric potential difference whose magnitude is close to the theoretical for ideal Ca2+ selectivity. The inhibitor of mitochondrial Ca2+ transport, ruthenium red, abolishes both the glycoprotein- and peptide-induced Ca2+ transport in bilayer lipid membranes. Thiol groups essential for Ca2+ transport activity were revealed in the glycoprotein and peptide. Addition of these substances to rat liver mitochondria induces Ca2+-dependent inhibition of the state 3 respiration that can be released by uncouplers (oligomycin-like effect).

Calcium binding sites with high and low affinity are revealed while only sites with high affinity to the calcium ion (Kd = 5.8 x 10(-6) M) are found in the glycoprotein component of the complex. A model of the glycoprotein-peptide complex functioning in the system of electrogenic transport of Ca2+ in mitochondria is suggested.

Glycoprotein (GP) inducing selective calcium transport through bilayer lipid membrane (BLM) from homogenate and mitochondria of beef heart was isolated by our method using no chaotrophic agents, detergents and proteolytic enzymes. 20-100 micrograms/ml of GP increase BLM, conductivity per 2-4 orders, producing potential close to the theoretical under ten fold gradient of calcium ions. M. W. of GP is about 40000 as determined by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Thiol groups are revealed in GP essential for Ca2+-transporting activity.

It is shown that glycoprotein from bovine heart mitochondria which forms Ca2+-selective conductance channels in a bilayer lipid membrane possesses Ca2+-binding activity. Ca2+-binding sites of two kinds were revealed in the glycoprotein molecule: high affinity sites with Kd = 2.8 X 10(-6) M and low affinity sites with Kd 1.1 X 10(-5) M. Ca2+-binding by the high affinity sites occurs co-operatively. The Hill coefficient is about 2.