CALIX[4]ARENES AS MODULATORS OF ENERGY-DEPENDENT CA2+_ACCUMULATION AND FUNCTIONING OF THE ELECTRON TRANSPORT CHAIN IN SMOOTH MUSCLE MITOCHONDRIA.

The influence of supramolecular macrocyclic compounds calix[4]arenes (C-97, C-99, C-107) at a concentration of 100 nM in the process of energy-dependent Ca²âº-transport in isolated mitochondria of smooth muscle, as well as autofluorescence mitochondrial coenzyme NADH, FAD and hydrodynamic diameter of these organelles was investigated. Using Ca²âº-sensitive fluorescent dye Fluo-4 AM it was shown that the selected calix[4]arenes can suppress energy-dependent accumulation of Ca²âº by mitochondria. Accumulation of Ca²âº (80 jiM in the medium) accompanied by the growth of the fluorescent probe response from a conventional unit to a value of 1,57±0,04 (n=5). Calix[4]arenes C-97, C-99, C-107 falls fluorescent signal below the 0,88±0,08, 0,92±0,08 and 0,78±0,04 respectively. Thus, the selected calix[4]arenas lead to release of previously accumulated Ca2+ from mitochondria. Under the influence of C-97 and C-99 fluorescent signal from NADH reduced to -0,11±0,02 and -0,12±0,02, respectively, in relation to the reference value - -0,05±0,01 (n=5). Analysis of fluorescence response NADH and FAD in a suspension of isolated mitochondria suggests that the effects of test compounds on the functional activity of the electron transport chain is associated with the initial stimulation of its 1-th complex and subsequent inhibition of Ca²âº-dependent NAD- containing Krebs cycle dehydrogenases. Along with this, the use of photon correlation spectroscopy to assess changes in the volume of mitochondria (their hydrodynamic diameter) under the action of selected calix[4]arenes has shown that interference with the electron transport chain leads to changes in the osmotic balance between the matrix of the mitochondria and the external environment. The result is the growth of isolated organelles volume. In particular, the hydrodynamic diameter of mitochondria increased by 22±6 % and 34±8 % (n=5) in presence of C-97 or C-99. The conclusion was done about the advisability of further studies of the calyx[4]arenes effect on smooth muscle Ca²âº-homeostase and mitochondrial bioenergetics in order to find effective modifiers of their func- tional activity.

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 µM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 µg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 µM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

ELECTROCHEMICAL POTENTIAL OF THE INNER MITOCHONDRIAL MEMBRANE AND Ca2+ HOMEOSTASIS OF MYOMETRIUM CELLS.

We demonstrated using Ca(2+)-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes' inner mitochondrial membrane by aprotonophore carbonyl cyanide m-chlorophenyl hydrazone (10 µM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3'-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane's electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hydrazone causes substantial increase in fluorescence of Ca(2+)-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca(2+)-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.

[Ca2+/H(+)-exchange in myometrium mitochondria].

Using the fluorescent probe Fluo-4 AM the authors have identified Na(+)-independent Ca2+H(+)-exchange in isolated mitochondria of rat myometrium and studied its individual properties. Formation of directional protons gradient in the matrix of mitochondria causes antyporte release of Ca2+, which has been previously accumulated in energetic processes (in the presence of Mg-ATP and succinate). The functioning of Ca2+/H(+)-exchange depends on the proton gradient and is characterized by reversibility, in case of extramitochondria environment alkalization the additional accumulation of Ca2+ by organelles is recorded. Monovalent cations gradients (Na+, K+, Li+) do not cause the release of Ca2+ from mitochondria. Rate of Ca2+/H(+)-exchange is growing in terms of increasing deltapH on the mitochondria membrane and kinetics of deltapH-induced Ca2+ release from the matrix corresponds to the laws of first order reaction. Research of Ca2+/H(+)-exchange some properties in the myometrium mitochondria showed that the above transport process is of electrogenic nature, perhaps it is done in a 1: 1 stechiometry (Hill coefficient on H+ close to 1) and is able to adjust matrix Ca2+ concentration under physiological conditions (pH activation of about 6.9). Thus, in the inner membrane of the myometrium mitochondria the available system of the secondary active Ca(2+)-transport from the matrix of these organelles to myoplasm and the functioning of Ca2+/H(+)-exchanger may underlie this process.

[Nitric oxide as a possible regulator of energy-dependent Ca2+ transport in mitochondria of uterine smooth muscle].

The influence of the donor and the precursor of NO, namely 100 mM sodium nitroprusside and sodium nitrite on the energo-dependent Ca(2+)-transport in isolated mitochondria from rat myometrium was investigated. Changes in the mitochondrial matrix Ca(2+)-concentration was evaluated by spectrofluorimetry using Ca2+ sensitive probe Fluo-4 AM. Mg(2+)-ATP-dependent Ca(2+)-accumulation on mitochondria in the presence of succinate significantly stimulated by nitric oxide, in particular, 100 microM sodium nitroprusside amplified the transport by 1.6 times relative to its control values. NO effect becomes significant only when the incubation of mitochondria with the compounds was performed. Ca(2+)-accumulation in the presence of sodium nitroprusside effectively suppressed by protonophore (CCCP) and ruthenium red (10 microM). It was concluded that inner mitochondrial membrane Ca(2+)-uniporter stimulated by nitrogen oxide. Ca(2+)-accumulation in mitochondria in the presence of sodium nitroprusside was not sensitive to the action of a specific permeability transition pore inhibitor cyclosporine (5 microM). This data indicates that the role of permeability transition pore is less significant than Ca(2+)-uniporter in the processes of Ca(2+)-transport in mitochondria under the nitric oxide action. Thus, nitric oxide stimulates the energo-dependent Ca(2+)-accumulation by myometrium mitochondria mediated their inner membrane Ca(2+)-uniporter functioning.

[Investigation of nitrosactive compounds influence on polarization of the mitochondrial inner membrane in the rat uterus myocytes using potential sensitive fluorescent probe DiOC6(3)].

The effect of nitrosactive compounds (sodium nitroprusside and sodium nitrite) on the polarization level of the uterus myocytes inner mitochondrial membrane using the confocal laser microscopy and fluorescent probe potentialsensitive DiOC6(3) (3,3'-dihexyloxacarbocyanine) was ivestigated. Colocalisation of mitochondrial membranes specific fluorescent probes (MitoTracker Orange CM-H2TMRos, 10 - nonyl acridine orange and DiOC6(3)) was demonstrated. It was shown that sodium nitroprusside at 0.1 mM concentration caused a moderate decrease in mitochondrial transmembrane potential. That observation was confirmed by flow cytometry. Action efficiency of sodium nitrite in a similar concentration was significantly lower than that of sodium nitroprusside. It is shown that it was sodium nitroprusside which caused a slight swelling of the mitochondria. A possible protecting role of nitric oxide as to mitochondria was discussed.

[H+-Ca2+-exchanger in the myometrium mitochondria: modulation of exogenous and endogenous compounds].

The properties of ΔpH-induced Ca2+-transport from isolated rat myometrium mitochondria was investigated. Ca2+-accu- mulation was carried out in the presence of Mg-ATP2- and succinate. Transport of Ca2+ recorded using Ca2+-sensitive fluorescent probe Fluo-4 AM. It is shown that acidification of extramitochondrial medium is accompanied by stimulation of Ca2+ release from mitochondria. This process is insensitive to the tetraphenylphosphonium which is relatively specific Na+-Ca2+-exchanger inhibitor of mitochondrial inner membrane, but inhibited in the presence of monoclonal antibodies directed against the protein LETM1 (Anti-LETM1). LETM1 protein in some tissues is the molecular basis of the H+-Ca2+-exchanger functioning on mitochondria. It was found that the H+-Ca2+-exchanger is stimulated by 100 µM amiloride (diuretic) and inhibited by Mg ions in milimolar concentrations. The transport system was completely resistant to the action of nitric oxide (sodium nitroprusside and sodium nitrite), but was stimulated by macrocyclic compounds of Calixarenes (C-97 and C-99) in submicromolar concentrations. Thus, the mitochondria of rat myometrium probably not have a system of Na+-Ca2+-exchanger, and provide the maintenance of the matrix Ca2+-homeostasis with H+-Ca2+-exchanger. Since the transport system high affinity activated by Calixarenes, further investigation of the influence of these compounds on the transport process makes promising.

[Ca2+ accumulation study in isolated smooth muscle mitochondria using fluo-4 AM].

The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presence of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating. The apparent activation constant of Ca2+ accumulation is 53.9 +/- 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation me- dium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.

[Investigation of the changes in uterine myocytes size depending on contractile activity modulators by photon correlation spectroscopy].

By using photon correlation spectroscopy the effect of contractile activity modulators of smooth muscle to the size (diameter) of rats uterine muscle cells was investigated. Cells were studied by using laser confocal microscopy as objects mostly oval or nearly oval form. The diameter of myocytes, assessed by photon correlation spectroscopy, was amounted 7-10 microm which is Consistent with the results obtained by using laser confocal microscopy. It is shown that the myoconstricted agents as Ca2+ ionophore A-23187 (10 microM) and K(+)-channels inhibitors (tetraethylammonium and 4-aminopyridine in a concentration of 1 mM) leading to decrease the diameter of the cells by 22%, 12% and 24% in average respectively. The ouabain, that reduces contractile activity of smooth muscle, leads to increase this parameter up 23% from control. Thus, decreasing/increasing the diameter of uterine myocytes correlates with their well known influence on myometrium constriction/relaxation.

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

[Registration of K(+)-equilibrium potential in myometrium cell plasma membrane and study of its modulation of NO(x) and H2O2, using flow cytometry method].

Prospects of the use of flow cytometry analysis for investigation of forming K(+)- equilibrium membrane potential on the experimental model of myometrium plasma membrane vesicles in the presence of valinomycine using potential-sensitive probe of DiOC6(3). Transmembrane potential magnitude corresponds to magnitude by Nernst's equation. H2O2 and NO2-, probably, increase permeability of membrane for K+ and result in potential dissipation. Given effect is not shown for sodium nitroprusside. The obtained results confirm an assumption as to enhancing the passive transport for K+ through sarcolemma under the action of these substances, that can lead to membrane repolarisation and decline of the level of its excitability.

[Effect of nitric oxides and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase activity in myometrium sarcolemma].

The action of sodium nitroprusside, nitrite-anions and hydrogen peroxide on Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (Ca(2+)-independent) enzymatic activity in myometrium sarcolemma fraction is investigated. It is established, that 0.1 mM sodium nitroprusside and 10(-8)-10(-5) M nitrite-anions essentially reduce Ca2+, Mg(2+)-ATPase activity whereas Mg(2+)-ATPase proved to be absolutely resistant to them. At rather high concentration of nitrite-anions (0.1 mM) appreciable stimulation of Ca2+, Mg(2+)-ATPase was observed. Hydrogen peroxide (10(-8)-10(-4)), depending on the concentration suppressed both enzymes activity. However, Ca2+, Mg(2+)-ATPase proved to be more sensitive to the action of H2O2 (seeming K(i) = 0.42 +/- 0.1 microM), than Mg(2+)-ATPase (seeming K(i) = 3.1 +/- 0.9 microM). At presence of 1 mM ditiothreitole (a reducer of SH groups of the membrane surface) action of investigated substances considerably decreased. Reagents on carboxic- (dicyclogexilcarbodiimid) and amino- groups of the membrane (trinitrobenzolsulfonic acid) inhibited both Ca2+, Mg(2+)-ATPase, and Mg(2+)-ATPase activity in membrane fractions. In the presence of noted reagents sodium nitroprusside and nitrite-anions action was not almost shown. Hence, nitrogen oxide, nitrite-anions and hydrogen peroxide suppress Ca2+, Mg(2+)-ATPase and Mg(2+)-ATPase (only hydrogen peroxide) activity in the plasmatic membrane of myometrium cells, and this action can be connected with direct updating of superficial chemical groups of the membrane.

[Effect of ionic and colloid gold on ATP-hydrolase fermentative systems in the membrane of Bacillus sp. B4253 and Bacillus sp. B4851].

Accumulation of gold in cells of Bacillus sp. B4253 can be directly or indirectly connected with activity of bacteria plasma membrane basal Mg2+-ATPase. Therefore this work deals with a comparative analysis of kinetic properties of plasma membrane basal azide-resistant Mg2+-dependent ATP-hydrolase activity of B. sp. B4253 and B. sp. B4851 capable to gold accumulation and not capable to this process, accordingly. It is shown, that by a number of kinetic parameters - specific fermentative activity, initial speed of reaction of hydrolysis ATP (V0), Mixaelis constant (Km), the maximal initial speed by Mg2+ (V(Mg)) and by ATP (V(ATP)), optimum concentration of ATP ([ATP]opt), pHmax, sensitivity to action of the thapsigargine and eosine Y - bacteria membranes basal Mg2+-ATPase activity accumulating gold, and the bacteria not capable to this process, are identical. But by some parameters they differ: Mg2+-ATPase activity of membranes of the bacteria which do not accumulate gold, has three times greater affinity for Mg ions and smaller value [Mg]opt. The inhibition effect of ionic gold (10(-4)-3x10(-4) M) is shown on azide-sensitive (H+-ATPase) and azide-resistant (Mg2+-ATPase) components Mg2+-dependent ATP-hydrolase activity in fraction of plasma membranes of microorganisms Bacillus accumulating gold, and not capable to this process. Colloid gold (0.0002-4 microg/ml) stimulates activity of H+-ATPase and Mg2+-ATPase in a membrane of the bacteria accumulating gold 1.5-2 times, and does not influence activity of ATPases of a membrane of the bacteria which do. not accumulate gold.

[Identification and catalytic properties of Mg2+-dependent ATP-hydrolase of cytoplasmic membrane of Bacillus sp. B4253 capable of gold accumulation]. / Identyfikatsiia ta katalitychni vlastyvosti Mg2+-zalezhnoï ATP-hidrolazy tsytoplazmatychnoï membrany Bacillus sp. B4253, zdatnykh do nakopychennia zolota.

Ca2+-independent, Mg2+-dependent ATP-hydrolase fermentative activity consisting of two components--azide-sensitive and azide-resistant ones has been identified in cytoplasmic membrane of Bacillus sp. B4253 capable to gold accumulation in ionic and colloid forms. The authors have characterized properties of the azide-resistant component of ATP-hydrolase reaction: dynamics of accumulation of one of the reaction products--inorganic phosphate P(i); dependence of ATP hydrolysis rate on the membrane protein content; pH-dependence; sensitivity of ATP-hydrolase activity to the change of reagents (ATP, Mg2+) concentration, as well as to the effect of some specific and nonspecific inhibitors of ion-transporting Mg2+-dependent ATP-hydrolase systems (ouabain, tapsigargin, eocine Y, La ions). It is supposed that the obtained experimental data can be used for the following study of molecular and membrane mechanisms of gold accumulation in Bacillus sp. B4253.

[Kinetic regularities of the proceeding and possible reaction mechanism of Mg2+-dependent enzymatic hydrolysis of ATP in the fraction of plasmatic membranes of the smooth muscle]. / Kinetychni zakonomirnosti perebihu ta mozhlyvyi mekhanizm reaktsiï Mg2+-zalezhnoho fermentatyvnoho hidrolizu ATR u fraktsiï plazmatychnykh membran hladen'koho m'iaza.

Kinetic regularities of the reaction of Ca2+-independent Mg2+-dependent enzymatic hydrolysis of ATP catalyzed by the so-called "basal" Mg2+-ATPase localized in the plasmatic membrane of the uterus smooth-muscle cells have been studied using the methods of kinetic analysis performed under the equilibrium conditions. The analysis was based on the study of the concentration dependence of initial velocity of nucleoside triphosphate hydrolysis in EGTA-containing medium under the change of general concentrations of ATP [ATP]o and Mg2+[Mg2+]o in conditions of their equimolar ratio ([ATP]o/ [Mg2+]o)= 1; here the ratio between the concentrations of free reagents ([ATP4-]o/[Mg2+]o) was equal to 1.25. The obtained concentration dependence was interpreted in terms of two practically possible alternative mechanisms of Mg2+-dependent ATP-hydrolase enzymatic reaction. Mechanism I. Two separate independent centres of Mg ions and ATP binding by the enzymatic protein are supposed to exist, while Mg2+-dependent ATP-hydrolase enzymatic reaction proceeds independent of the equilibrium reaction of Mg ions chelatization of muscleside triphosphate. Mechanism II. The existence of the only centre of the chelate complex Mg2+ATP2- binding is postulated on the enzymatic protein; this process is also realized independent of the binding of Mg2+ and ATP-hydralase reaction catalized by it.

[Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes]. / Vplyv eozynu Y na Ca2+-nezalezhnu, Mg-zalezhnu ATP-aznu aktyvnist' plazmatychnykh membran hladen'kom'iazovykh klityn.

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.

[Identification and characterization of "basal" Ca2+-independent Mg2+-dependent ATP-hydrolase enzymatic activity in smooth muscle cell plasma membrane fractions]. / Identyfikatsiia ta kharakterystyka "bazal'noï" Ca2+-nezalezhnoï Mg2+-zalezhnoï ATP-hidrolaznoï fermentatyvnoï aktyvnosti u fraktsiï plazmatychnykh membran hladen'kom'iazovykh klityn.

It is shown, that for correct definition of "basal" Ca(2+)-independent Mg(2+)-dependent ATPase ac-activity (10-13 mmol Pi/hour on 1 mg of protein) in a fraction of uterus smooth muscle cell plasma membranes is necessary to use in medium without calcium of an incubation not only EGTA and digitonin--of the factor of infringement in activity by this subcellular structure, but inhibitors of others Mg(2+)-dependent ATP-hydrolyse enzymatic systems localized as in plasma membrane (Na+, K(+)-ATPase) and in others subcellular frames, first of all, in mitochondria (Mg(2+)-ATPase) and endoplasmic reticulum (transport Ca2+, Mg(2+)-ATPase). In the case of a sacolemal fraction of a smooth muscle the contribution of others Mg(2+)-dependent ATP-hydrolyse systems in a common enzymatic hydrolysis ATP, which unconnected to functioning "basal" Ca(2+)-independent Mg(2+)-dependent ATPase, is very appreciable and achieves 35%. The researches, carried out in the frameworks of definition of initial velocity of enzymatic reaction, have enabled to define its some properties--cationic and anionic specificity, and also sensitivity to action of some inhibitors. It has appeared, that the "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction is nonspecific rather both in relation to cations of divalent metals Me2+, and cations of monovalent metals and anions, which were utilized for support of ionic strength. The cations La--antagonist of cations Ca--practically did not influence enzymatic activity. The non-specific inhibitors transport of ATPases--p-chloromercuribenzoate, o-vanadate and eosine Y with a various degree of efficiency inhibited "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction. On the basis of the analysis of the own and literary data the conclusion is made that "basal" Ca(2+)-independent Mg(2+)-dependent ATPase of a smooth muscle cell plasma membrane is considerably less sensitive to action of nonspecific inhibitors of the Ca(2+)-transporting systems, than these systems.