[Ethanol sensitivity of rat brain cortex Na(+), K(+)-ATPase in plasma membrane structural damage induced by sodium dodecyl sulfate]. / Chuvstvitel'nost' Na(+), K(+)-ATP-azy kory golovnogo mozga krys k étanolu pri narushenii struktury plazmaticheskoi membrany dodetsilsul'fatom natriia.

To investigate the role of rat brain cortex Na+, K(+)-ATPase plasma membrane microenvironment in ethanol effect in vitro on membrane the sensitivity of enzyme activity to alcohol was studied under membrane perturbation induced by sodium dodecyl sulfate. The increase of enzyme sensitivity to detergent inactivation in the presence of high ethanol concentrations and to alcohol inhibition after modification by Ds-Na was revealed. It is supposed that Na+, K(+)-ATPase sensitivity to ethanol is dependent on structural state of protein microenvironment in accordance with assumed differences in structural organization of the boundary lipids of the neuronal enzyme isoforms.

[Effect of inhibitors of energy-dependent Ca2+-transporting systems on ATP-hydrolase activity of smooth muscle actomyosin]. / Vliianie ingibitorov énergozavisimykh Ca2+-transportiruiushikh sistem na ATP-gidrolaznuiu aktivnost' aktomiozina gladkoi myshtsy.

Comparative investigation of of smooth muscle actomyosine ATP-ase sensitivity to some inhibitors of energy-dependent Ca(2+)-transporting systems has been carried out. It is proved that the ATP-ase of actomyosine is nonselectively inhibited by thapsigargin (imaginary inhibition constant Ki is equal 29.4 +/- 5.2 nM), cyclopiazonic acid (Ki = 626 +/- 118 nM), eosin Y (Ki = 70 +/- 14 nM) and p-chlormercurybenzoate (Ki = 380 +/- 151 nM). The data obtained could be used for the further development of the ideas about regularities of Ca(2+)-dependent control of the smooth muscles contraction-relaxation.

Effects of eosin Y on the catalytic and functional activities of Mg2+,ATP-dependent calcium pump of smooth muscle cell plasma membrane.

Experiments with highly purified preparations of Ca2+,Mg2+-ATPase solubilized from sarcolemma and a fraction of inside-out sarcolemmal vesicles were performed to study the kinetics of inhibitory effects of eosin Y (0-100 microM) on the catalytic and transport activity of Mg2+,ATP-dependent calcium pump of myometrial cell plasma membrane. For both the Ca2+,Mg2+-dependent ATP hydrolysis and the Mg2+, ATP-dependent accumulation of Ca2+ the apparent inhibitory constant Ki was 0.8 microM. However, eosin Y used at concentrations of up to 100 microM had absolutely no effect on Na+-Ca2+ exchange in the plasma membrane fraction. This inhibitor decreased the turnover rate of purified Ca2+,Mg2+-ATPase determined both by Mg2+ and ATP. However, the affinity of purified Ca2+,Mg2+-ATPase for Mg2+ increased, and its affinity for ATP decreased in the presence of eosin Y. In the case of Mg2+,ATP-dependent Ca2+ transport, eosin Y decreased the turnover rate of the calcium pump whose affinity for Mg2+ and ATP displayed virtually no dependence on the presence of the inhibitor in the incubation medium. The possibility of the use of eosin Y in model experiments for identification of Mg2+, ATP-dependent and Na+-dependent calcium fluxes from smooth muscle cells into the intercellular milieu is discussed.

[Effect of organic solvents on catalytic and functional activity of transport Ca2+,Mg2+-ATPase from smooth muscle cell membrane]. / Vliianie organicheskikh rastvoritelei na kataliticheskuiu i funktsional'nuiu aktivnost' transportnoi Ca2+-Mg2+-ATFazy plazmaticheskoi membrany gladkomyshechnykh kletok.

It was shown that organic solvents (dioxane, acetone, ethanol, dimethylsulfoxide) at concentrations of < 10% suppress the activity of transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membranes of smooth muscle cells and Mg(2+)-ATP-dependent accumulation of Ca2+ ions in inverted membrane vesicles. It was found that one of the reasons for the inhibition of enzymatic and transport activity of Ca2+, Mg(2+)-ATPase by the action of these solvents is an increase in the attractive force between oppositely charged active center of the enzyme and the product (products) of the ATP-hydrolase reaction, which is induced by a decrease in the dielectric permeability of incubation medium.

[Effect of vitamin D3 and 20-hydroxyecdysone on the content of ATP, creatine phosphate, carnosine and Ca2+ in skeletal muscles]. / Vliianie vitamina D3 i 20-gidroksiékdizona na soderzhanie ATP, kreatinfosfata, karnozina i Ca2+ v skeletnykh myshtsakh.

The effect of vitamin D3, 20-hydroxyecdysone and extract from Serratula coronata containing 20-hydroxyecdysone on the level of basic metabolites in the skeletal muscles of rats has been studied. It was shown that development of D-hypovitaminosis is accompanied by the decrease in content of ATP, creatine phosphate, carnosine, and by the increase of Ca2+ content. Against the background of D-hypovitaminosis the 20-hydroxyecdysone and the extract from Serratula coronata which contains 20-hydroxyecdysone promote the increase of the amount of these metabolites up to the control of one and normalize Ca2+ content in them.

[Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane]. / Kineticheskie zakonomernosti reaktsii gidroliza ATP, kataliziruemoi Mg2+-ATPazoi plazmaticheskoi membrany gladkomyshechnykh kletok.

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.

[Identification of membrane proteins of sarcoplasmic reticulum binding 2,4,6-trinitrobenzene sulfonate]. / Identifikatsiia belkov membran sarkoplazmaticheskogo retikuluma, sviazyvasiushchikh 2,4,6-trinitrobenzolsul'fonat.

Sarcoplasmic reticulum was treated with 2,4,6-trinitrobenzene sulfonate (TNBS), which binds covalently only with surface amino groups. After solubilization of the membranes and gel-filtration of the SR protein on the Sephadex G-100, the label was discovered in the protein fraction 100-80 and 70-60 kDa. The amino groups of proteins with molecular weight 50-20 kDa are inaccessible for the 2,4,6-TNBS. Incorporation of the latter into proteins increases in presence of KCl.

[Passive Ca2+ influx into vesicles of the sarcoplasmic reticulum, modified by succinic anhydride]. / Passivnyi vkhod Ca2+ v vezikuly sarkoplazmaticheskogo retikuluma, modifitsirovannye iantarnym angidridom.

Treatment of the sarcoplasmic reticulum (SR) vesicles with succinic anhydride in concentration of 1-2 mM modifies about 20% of amino groups. It increases initial rate and changes the pH-dependence of the passive influx of Ca2+ into vesicles and does not affect either Ca(2+)-binding or maximal passive Ca(2+)-loading of the SR vesicles. It is supposed that this effect may be caused by modification of the Ca-channel gating behaviour as a result of replacement of positive surface amino groups by carboxyl groups.

[Change in the passive influx of Ca2+ into sarcoplasmic reticulum vesicles as a result of chemical modification of surface amino groups]. / Izmenenie passivnogo vkhoda Ca2+ v vezikuly sarkoplazmaticheskogo retikuluma v rezul'tate khimicheskoi modifikatsii poverkhnostnykh aminogrupp.

Treatment of sarcoplasmic reticulum (SR) vesicles with trinitrobenzene (TNBS) and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) stimulates the initial rate of passive influx of Ca2+ into SR vesicles, but does not affect either the binding or the maximal passive loading of SR vesicles with Ca2+. The changes in the kinetics of KCl-stimulated passive influx of Ca2+ depend on the reagent used. It is supposed that stimulation of passive influx of Ca2+ into SR vesicles and the changes in the reaction kinetics may be caused by modification of the Ca2+ channel gating behaviour as a result of binding of surface amino groups.

[Calcium ion currents in the smooth muscle of the myometrium]. / Potoki ionov kal'tsiia v gladkoi myshtse miometriia.

The results of kinetic analysis of Ca liberation from preparations of female rabbit myometrium show that in the uterus smooth muscle there exist three pools of cation with characteristic times of metabolism 182.18 +/- 25.20, 25.56 +/- 1.00 and 2.94 +/- 0.38 min respectively. It was concluded from the compartmentalization analysis and from the data on Na+- and Mg2+--ATP-dependent transport in plasmic membrane fraction of myometrium cells that the fast phase of calcium metabolism reflects the liberation of the cation from extracellular space in the incubation medium, the intermediate one--Ca transfer from myocytes into the extracellular medium, and the slow one--liberation from the subcellular structures into the myoplasm.

[Levels and intracellular distribution of calcium and magnesium in the myometrium]. / Soderzhanie i vnutrikletochnoe raspredelenie kal'tsiia i magniia v miometrii.

Atom-absorption spectrophotometry have shown that the content of Ca2+ in the rabbit and cow myometrium amounts to 4.54 +/- 0.47 and 2.57 +/- 0.30 and that of Mg2+--3.89 +/- 0.15 and 1.35 +/- 0.17 mmol per 1 kg of wet tissue weight, respectively, The content of Mg2+ in the myometrium is two times lower than in the myocardium and three times lower than in the skeletal muscle. During pregnancy (the day before delivery), delivery and postdelivery period the Ca2+ content in the rabbit myometrium is 1.5-2 times lower than in the state of functional rest, and its specific content in fractions of nuclei, mitochondria, microsomal and plasma membranes is practically the same (100-140 nmol per 1 mg of fraction protein). Distribution of the total Ca content calculated per fraction protein satisfies the following series: soluble fraction (56.4%) greater than nuclei (23.6% greater than mitochondria (7.4%) greater than microsomes (1.9%) greater than or equal to plasma membranes (1.3%). The highest specific content of Mg2+ is observed in the fraction of: plasma membranes--52, then mitochondria--40, microsomes--27 and nuclei--19 nmol per 1 mg of protein. The distribution of the total content of this element is described by a series: soluble fraction (71.8%) greater than nuclei (8.3%) greater than mitochondria (4.6%) greater than plasma membranes (1.7%) greater than microsomes (0.4%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Contribution of the Mg2+-, ATP- AND Na+-dependent Ca2+-transport systems to the regulation of Ca2+ concentration in myometrial cells]. / Vklad sistem Mg2+, ATP- i Na+-zavisimogo transporta Ca2+ v reguliatsiiu ego kontsentratsii v kletkakh miometriia.

Studies with sarcolemma from cattle myometrium containing inside-out cytoplasmic vesicles, using Ca2+-EGTA buffer, showed that the affinity of ionized Ca2+ for the Mg2+- or ATP-dependent transport is higher than that for the Na+-Ca2+ exchange system (Kd = 3,2 X 10(-6) and (4.3-5.3) X 10(-5) M), respectively. The Km values for MgATP are 2.15 mM. Oxytocin added to the homogenization medium containing rabbit and cattle myometrium cells, i.e. during the formation of closed sarcolemmal fragments, resulted in inhibition of Mg2+, ATP-dependent accumulation of 45Ca2+ by plasma membranes. However, an addition of oxytocin to the incubation medium did not affect the kinetics of active accumulation of Ca2+. It was assumed that the system of non-electrogenic Na+-Ca2+ exchange in the myometrium possessing a low affinity for Ca2+ provides for the maintenance of ionized Ca2+ concentration in the myocytes at 10(-5) M. Therefore, this system cannot induce relaxation of mechanical tension of the uterus. Further decrease of Ca2+ in the myoplasm from 10(-5) to 10(-7) M and, correspondingly, the relaxation of myometrium is provided for by the Mg2+, ATP-dependent efflux of Ca2+ from the myocytes having a high affinity for this cation. The decrease of the activity of ATP-dependent Ca2+-pump by oxytocin is the cause of Ca2+ elevation in the myoplasm and, consequently, of myometrium contraction.

[ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium]. / ATP-zavisimoe nakoplenie Ca2+ fraktsiei plazmaticheskikh membran miometriia.

The specific activities of Mg2+, Ca2+-ATPase in the plasma membrane fraction of rabbit and cattle myometrium are 8.30 +/- 0.80 and 2.36 +/- 0.48 mkmoles of Pi per mg of protein, respectively. This fraction possesses a higher (in comparison with other subcellular fractions) capacity for ATP-dependent uptake of 45Ca2+ (9.37 +/- 1.66 and 6.86 +/- 0.96 nmoles of 45Ca2+ per mg of protein in 15 min for rabbit and cattle myometrium, respectively); the ratio of ATP-dependent uptake of Ca2+ to adsorbed Ca2+ is also high. Phosphate increases Ca2+ uptake in the presence of ATP and Mg2+. The ionophore A-23187 added to the incubation mixture without ATP and Mg2+ sharply increases Ca2+ binding. An addition of the ionophore at the 15th min of the ATP-dependent Ca2+ uptake causes a complete and rapid release of the accumulated Ca2+. The release of Ca2+ can be also caused by an addition of Na-DS or EGTA to the incubation mixture. This suggests that Ca2+ is accumulated through the plasma membrane inside the closed structures. It was assumed that myometrial sarcolemma plays an essential role in regulation of intracellular Ca2+ concentration in the uterus at rest and that the active Ca2+ efflux from the cells is controlled by the Mg2+, Ca2+-ATPase system.