[Phosphorylation of rat brain synaptosomal proteins stimulated by alpha-latrotroxin]. / Fosforilirovanie belkov sinaptosom golovnogo mozga krys, stimuliruemoe alph-latrotoksinom.

alpha-Latrotoxin (LTX) is a presynaptic neurotoxin from black widow spider (Latrodectus mactans tredecimguttatus) venom; it causes massive Ca(2+)-independent neurotransmitter release. The effect of LTX on phosphorylation of synaptosomal proteins was studied including synapsin I, synaptotagmin, and dynamin. Experiments were performed in Ca(2+)-supplemented medium containing 1 mM CaCl2 or nominally Ca(2+)-free medium (without added Ca2+ and EGTA) using intact synaptosomes isolated from rat brain and prelabeled with 32P(i). The data indicate the exposure of synaptosomes to 10 nM LTX for 10 sec stimulates 32P(i) incorporation into synapsin I up to 143% versus control in Ca(2+)-supplemented medium and up to 130% in Ca(2+)-free medium. LTX (20 nM) significantly stimulated synapsin I phosphorylation (up to 173% versus control) only in Ca(2+)-free medium. Under these conditions, dephosphorylation of dynamin was not observed. Exposure of synaptosomes to LTX in Ca(2+)-supplemented medium for 10 sec did not affect 32P(i) incorporation into synaptotagmin but increase in incubation time up to 30 sec results in dephosphorylation of synaptotagmin down to 70% versus control level. In Ca(2+)-free medium, LTX stimulates the 32Pi incorporation into synaptotagmin (up to 140% versus control) and the effect was not time-dependent. Thus, LTX-stimulated neurosecretion in Ca(2+)-supplemented and Ca(2+)-free medium requires phosphorylation of synapsin I. Phosphorylation-dephosphorylation cycle of synaptotagmin can be important for regulation of recyclization of synaptic vesicles.

[Functioning of latrotoxin channels during pH changes]. / Funktsionirovanie latrotoksinovykh kanalov pri izmenenii pH.

Using the fluorescent probe BCECP, the pH dependence of Ca2+ transport in synaptosomes along alpha-latrotoxin-formed channels, was studied. It was found that the pH value in synaptosomes is equivalent to 7.16 +/- 0.09. Acidification or alkalinization of the intracellular medium by 0.1-0.3 pH units had no appreciable influence on the Ca2+ influx along latrotoxin-formed channels. Alteration of external pH caused a parallel shift in the cytoplasmic pH in the synaptosomes. The pH decrease in the external medium down to 6.0 caused the inhibition of Ca2+ fluxes along latrotoxin-formed channels. Dissipation of the proton gradient by high concentrations of KCl in the presence of nigericin decreased the latrotoxin ability to form ionic channels without any loss in the activity of the preformed channels. The influx of bivalent cations along latrotoxin-formed channels led to alkalinization of the synaptosomal cytoplasm to pH of the external medium. This pH change did not depend on the presence of Na+ in the external medium and was blocked by cadmium.

[Immunochemical and functional study of the latrotoxin molecule]. / Immunokhimicheskoe i funktsional'noe issledovanie molekuly latrotoksina.

A panel of monoclonal antibodies (mAb) against alpha-latrotoxin (LT) has been produced and their main characteristics have been determined. The influence of mAb on the functional effects of LT in synaptosomes from rat brain and on the channel formation in bilayer lipid membrane has been investigated. These mAbs do not inhibit binding of LT to rat synaptosomes but modify LT-receptor interaction in terms of LT's channel-forming and secretogenic effects. Antibodies A6 and A24 block these effects, whereas A4 partially preserves the secretogenic action of LT and completely blocks its channel-forming action. Only antibodies A15 affect the LT ability to form cationic channels in BLM, inducing considerable decrease in the frequency of the channel formation. These data and their analysis allow to identify several functional (and, probably, structural) domains of LT responsible for: 1) toxin-receptor interaction; 2) channel-forming and related calcium-dependent secretogenic effects; 3) calcium-independent secretogenic effects; 4) formation of cationic channels in the artificial lipid bilayer.

[A high level of cytoplasmic calcium inactivates ion channels, formed by alpha-latrotoxin in the rat brain synaptosomes]. / Vysokii uroven' tsitoplazmaticheskogo kal'tsiia inaktiviruet ionnye kanaly, formiruemye alpha-latrotoksinom v sinaptosomakh mozga krys.

Effect of alpha-latrotoxin on the concentration level of free calcium [( Ca2+]in) in the rat brain synaptosomes and dependence of the activity of "latrotoxin" channels on [Ca2+]in were studied using fluorescent calcium probe quin-2. It is shown that alpha-latrotoxin exerts effect on calcium permeability of plasmalemma and does not induce calcium ejection from the intracellular compartments. A lag-period is characteristic of alpha-latrotoxin action. A degree of the [Ca2+]in increase in synaptosomes depends on the toxin concentration. When [Ca2+]in increases as a result of preliminary potassium depolarization of plasmalemma of synaptosomes, the amount of incoming calcium ions followed by the toxin effect as well as the calcium input rate considerably decrease. Inactivation of calcium-transferring channels induced by alpha-latrotoxin is not a result of a change in the potential on the membrane, as during the blockage of potential-depending calcium channels by D-600, an increase of KCl in the incubation medium does not influence the alpha-latrotoxin action. Differences in the properties of alpha-latrotoxin channels are discussed in synaptosomes and BLM.

[Internalization and degradation of latrotoxin bound to rat brain synaptosomes]. / Internalizatsiia i degradatsiia latrotoksina, sviazannogo s sinaptosomami golovnogo mozga krys.

The accessibility to trypsin of 125I-labeled latrotoxin bound to rat brain synaptosomes was investigated. It was shown that latrotoxin bound to synaptosomes in the cold can be practically completely removed by trypsin treatment. The resistance of latrotoxin to proteolysis increases during its incubation with synaptosomes (37 degrees C). Concanavalin A (10(-6) M) decreases toxin binding by 30%, but fully prevents internalization (incorporation). Moreover, latrotoxin is not incorporated into synaptosomal membrane fragments irrespective of duration and temperature of incubation. Latrotoxin incorporated into synaptosomal membranes undergoes degradation by endogenous proteases resulting in the formation of TCA-soluble products.

[Binding of latrotoxin to synaptosomes and synaptic membranes of the rat brain]. / Sviazyvanie latrotoksina s sinaptosomami i sinaptocomal'nymi membranami mozga krys.

The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.

[ATP level in synaptosomes--a determining factor of the functional activity of ion channels induced by alpha-latrotoxin]. / Uroven' ATP v sinaptosomakh--opredeliaiushchii faktor funktsional'noi aktivnosti ionnykh kanalov, indutsiruemykh alpha-latrotoksinom.

The paper deals with characteristics of relationship between synaptosomal calcium permeability induced by alpha-latrotoxin and cytosolic concentration of ATP. It is shown that reagents decreasing the ATP level in the synaptosomes (monoiodoacetate, papaverine) inactivate the toxin-induced ionic fluxes and, on the contrary, reagents increasing the ATP level in synaptosomes enhance the toxin-induced calcium influx. The treatment of synaptosomes with inhibitors of phosphodiesterase of cAMP and cAMP-dependent protein kinase has no effect on the alpha-latrotoxin-induced calcium influx.

[Interaction of alpha-latrotoxin with synaptosomes; metabolic control of the induction of calcium channels]. / Vzaimodeistvie alpha-latrotoksina s sinaptosomami; metabolicheskii kontrol' induktsii kal'tsievykh potokov.

The aim of the present study is to elucidate the possible significance of metabolic control of the calcium permeability of synaptosomes induced by alpha-latrotoxin. The alpha-latrotoxin influence of processes of GABA release and reuptake is shown to depend on synaptosomal metabolism. It has been found that incubation by the synaptosomes during one hour at 37 degrees C or their treatment by 10(-3) M monoiodacetate during 10 min at 10 degrees C or at room temperature completely abolished or at least strongly decreased the ability of alpha-latrotoxin to induce the calcium influx. Under the same conditions the ability of alpha-latrotoxin to activate the GABA release and inhibit the GABA reuptake do not change. It is concluded that only formation of calcium channels by alpha-latrotoxin is strictly controlled by the level of synaptosomal metabolism.

[Calcium permeability of synaptosomes induced by alpha-latrotoxin]. / O kal'tsievoi pronitsaemosti sinaptosom, indutsirovannoi alpha-latrotoksinom.

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.

[Transmembrane localization of cytosol proteins from the heart muscle in proteoliposomes]. / O transmembrannoi lokalizatsii belkov tsitozolia serdechnoi myshtsy v proteoliposomakh.

It was shown that soluble cytoplasmic proteins of rabbit cardiac muscle were able to penetrate into phospholipid bilayer during preincubation with liposomes. The incorporation of cytoplasmic proteins into the liposomic membrane induced the sodium permeability that was sensitive to neurotoxins (veratridine and tetrodotoxin). This effect was partly eliminated by addition of tetrodotoxin to the external medium and completely eliminated in the presence of tetrodotoxin on the both sides of the liposomic membrane. It was found also that some of the proteins incorporated into the liposomes become resistant to pronase digestion when the enzyme is added to external medium. At the same time pronase included in the internal volume of the proteoliposomes was able to digest partly proteoliposomic proteins. All these data support the ideas of the integral incorporation of soluble cytoplasmic proteins into the liposomic membrane and to the existence of cytoplasmic precursor of voltage-dependent sodium channel.

[Biochemical studies of potential-dependent sodium channels]. / Biokhimicheskie issledovaniia potentsialzavisimykh natrievykh kanalov.

The data on interaction of sodium channels of excited membranes with various neurotoxin groups are considered in the paper including the data on using biologically active neurotoxin derivatives as a specific label. These derivatives carry fluorescent or photoactive groups possessing high specific radioactivity, which permits developing the methods for sodium channel solubilization and solubilized preparation stabilization. The high-purified preparations are characterized by the methods of gel chromatography, centrifugation within the gradient density, gel electrophoresis and affinity chromatography. Experiments on reconstruction of solubilized preparations in the bilayer of lipid vesicles are under discussion.

[Interaction of tetrodotoxin-sensitive structures of soluble cardiac muscle proteins with liposomes]. / O vzaimodeistvii tetrodotoksin-chuvstvitel'nykh struktur rastvorimykh belkov serdechnoi myshtsy s liposomami.

It is studied how conditions of soluble myocardium proteins interaction with liposomes provide formation of the channel-like structures with tetrodotoxin- and veratrine-dependent sodium permeability. The temperature is shown to influence the formation rate of neurotoxin-sensitive proteoliposomes. The veratrine effect on sodium permeability of proteoliposomes intensifies with the rise of temperature. Pretreatment of liposomes by veratrine prevents formation of tetrodotoxin sensitivity in proteoliposomes.

[Effects of neutral salts and temperature on skeletal muscle sarcolemmal Ca-TPase]. / Vliianie neitral'nykh solei i temperatury na Ca-ATPazu sarkolemmy skeletnykh myshts.

The temperature dependence and effects of sodium and potassium chloride on purified preparations of sarcolemmal Ca2+-activated ATPase were investigated. It was shown that within the concentration range of 0,1--1,0 M both salts have the same effect on the enzyme activity. A low ionic strength and concentration of the salts of 0,1 M the temperature maximum was 45 degrees and the shapes of temperature curves were the same. The Arrhenius plots showed a break at 16--19 degrees. The apparent activation energies were 27,3 kcal/mole below and 17,1 kcal/mole above the break point. At high ionic strength (0,5 M) the temperature maximum was observed at 40 degrees and the apparent activation energies decreased down to 18,0 kcal/mole below and 11,5 kcal/mole above the break point.

[Purification and some properties of skeletal muscle sarcolemma Ca2+-ATPase]. / Ochistka i nekotorye svoistva Ca-ATPazy sarkolemmy skeletnykh myshts.

Ca2+-ATPase of skeletal muscle sarcolemma has been isolated and purified. It is prepared from salt extract of sarcolemma by ammonium sulfate fractionation and further purified by gel chromatography on Sepharose 4B. The purity of preparations was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has been shown that Ca2+-ATPase possesses the same mobility as skeletal muscle myosin under gel chromatography on Sepharose 4B and the same mobility as myosin heavy chains in sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Membrane protein binds to rabbit skeletal muscle actin, and this complex dissociates by ATP. Interaction with actin does not change Ca2+- or Mg2+-stimulated ATPase activity. Enzyme has only one pH optimum at 7,0-7,6. Membrane protein is highly specified to calcium--ATPase activity in the presence of Mn2+ is 10% and in the presence of Sr2+, Mg2+ or Co2+ are 3-5% of the activity in the presence of Ca2+. Other nucleoside triphosphate (UTP and ITP) are hydrolyzed at lower rates than is ATP.

[Kinetic properties of skeletal muscle sarcolemmal Ca2+-ATPase]. / O kineticheskikh svoistvakh Ca2+-ATPazy sarkolemmy skeletnykh myshts

Calcium activation of skeletal muscle sarcolemma Ca2+-ATPase is investigated. The investigation of a dependency of the initial rate of ATP hydrolysis on total concentration of substrate and on total and free calcium concentrations showed that the role of calcium ions is not limited by the formation of the substrate complex (CaATP2-). Calcium is absolutely necessary for the enzyme transition from inactive into active form. The inhibitory effect of free ATP is due to a decrease of free calcium concentration as a result of complexation with ATP, but not of competition with substrate in the active site. It is shown also that magnesium competitively inhibits the interaction of the enzyme with the substrate and non-competively suppress the activation of Ca2+-ATPase by free calcium.

[Effects of magnesium, manganese and cobalt ions on the activity of Ca2+-dependent ATPase of skeletal muscle plasma membranes]. / Vliianie magniia, margantsa i kobal'ta na Ca2+-zabisimuiu ATRazu plazmaticheskikh membran skeletnykh myshts

Bivalent cations (Mn and Co) are found to activate ATPase activity of sarcolemma in the same degree, as Mg and Ca. The increase of the activity of Ca2+-dependent ATPase under the certain conditions of treatment of sarcolemma with KCl-Triton X-100 solution is not accompanied by the increase of ATPase activity in the presence of Mn and Co ions. ATPase activities in the presence of these ions correlate with the activity of Mg2+-dependent ATPase. Bivalent cations Mg, Mn and Co are competitive inhibitors for Ca2+-dependent ATPase. The apparent inhibition constants are determined to be 3,5-10(-5) M for Mg, 0,7-10(-3) M for Co and 1,5-10(-3) M for Mn. It is supposed that Ca2+-dependent ATPase has similar selectivity to bivalent cations as calcium influx that follows depolartization of plasma membrane.