[Mechanisms of Ca2+ transport to the cytoplasm of muscle cells, role of protons and active nitrogen (oxygen) metabolites in these processes].

Separate pictures of transmission mechanisms of Ca(2+)-signal are discussed, and first of all in the muscle cells. Literary and own data about possible interrelation of Ca2+ and H+ metabolism are analysed. Supposition about the important role of H+ on the first stages of activating the muscle cells is expressed. The role of active nitrogen and oxygen metabolites are discussed.

[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.

[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.

[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.

[The effect of pH on the passive transport of Ca2+ by membranes of fragmented sarcoplasmic reticulum of skeletal muscles]. / Vliianie pH na passivnyi transport Ca2+ membranami fragmentirovannogo sarkoplazmaticheskogo retikuluma skeletnykh myshts.

The initial rate of Ca2+ translocation in vesicular preparations of the sarcoplasmic reticulum membranes is shown to fall with a pH decrease to 6.0 or 5.0 and to rise with a pH change to 7.0 to 7.8 in respect to the initial 6.5. It is established that the Ca2+ sorption by the membranes or their fluidity make no essential contribution to the recorded changes of 45Ca2+ level in the membrane preparations. It is shown that the passive Ca2+ transport depends to a considerable extent on the concentration of a proton at the outer surface of the sarcoplasmic reticulum membrane: an excess of H+ inhibits the Ca2+ input and output, while a decrease of the proton concentration promotes an increase in the rate of these processes in the sarcoplasmic reticulum.

[Isolation of Ca 2+-activated photoprotein obelin from Obelia longissima and its use for the recording the outflow of Ca 2+ from fragmented sarcoplasmic reticulum of skeletal muscles]. / Poluchenie Ca 2+-aktiviruemogo fotoproteina obelina iz Obelia longissima i ego primenenie dlia registratsii vykhoda Ca 2+ iz fragmentirovannogo sarkoplazmaticheskogo retikuluma skeletnykh myshts.

A high-active stable preparation of obelin has been obtained from the luminescent hydroid Obelia longissima. The preparation is appropriate for determining free Ca2+ in the physiological range of its concentrations Obelin is shown possible to be used to record the processes of Ca2+ release from vesicles of the sarcoplasmic reticulum. In this case a rapid initial phase of Ca2+ outflux replaced by a slower one has been registered. A sharp increase of luminescence caused by the appearance of free Ca2+ in the medium has been registered under the effect of agents either increasing permeability of sarcoplasmic reticulum membranes for Ca2+ (A23187) or destroying the membrane (ethanol, triton X-100). The observed effects are confirmed, a radioactive label being used.

[Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH]. / Vykhod Ca 2+ iz vezikul sarkoplazmaticheskogo retikuluma pri izmenenii membrannogo potentsiala, kontsentratsii Ca2+ i pH.

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.

[A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. I. Passive Ca2+ efflux from vesicle membranes]. / Issledovanie passivnykh potokov Ca2+ cherez membranu sarkoplazmaticheskogo retikuluma skeletnykh myshts. I. Passivnyi vykhod Ca2+ iz membrannykh vezikul.

The same level of passively loaded Ca2+ was observed both in the heavy (enriched in terminal cisternae) and light (enriched in longitudinal reticulum) sarcoplasmic reticulum (SR) fractions. The level of passively loaded Ca2+ of the both SR fractions decreased in the presence of 150 mM K+. However the rate and extent of Ca2+ release was greater from heavy SR fraction. The rate of Ca2+ release under conditions of antiport of K+, Na+, choline+ and gluconate-, Cl-, SCH- increased proportion with their permeability through the SR membrane. The initial rate of Ca2+ release also became higher under equal concentration of monovalent cation chloride both inside and outside the SR vesicles. Apparently, in this case Ca2+ release occurs through Ca-channels which are open at a membrane potential.

[A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. II. Stimulation of passive Ca2+ influx with monovalent cations and anions]. / Issledovanie passivnykh potokov Ca2+ cherez membranu sarkoplazmaticheskogo retikuluma skeletnykh myshts. II. Stimuliatsiia passivnogo vkhoda Ca2+ odnovalentnymi kationami i anionami.

The initial rate of passive Ca2+ influx into "heavy" and "light" fractions of sarcoplasmic reticulum (SR) vesicles increases in the presence of univalent cation chlorides. Stimulation of passive Ca2+ influx decreases in the following order: KCl + valinomycin-KSCN- + valinomycin greater than KSI = NaCl greater than choline chloride. K-gluconate + valinomycin and K-gluconate have no effect on the passive Ca2+ influx into SR vesicles. It is supposed that KCl-stimulation of passive Ca2+ influx into SR vesicles under conditions used may be caused by depolarization of the SR membrane.

[The role of Ca2+-ATpase and its hydrophobic component in the release of Ca2+ from skeletal muscle sarcoplasmic reticulum]. / Uchastie Ca2+-ATPazy i ee gidrofobnogo fragmenta v osvobozhdenii Ca2+ iz sarkoplazmaticheskogo retikuluma skeletnykh myshts.

The Ca2+ permeability of proteoliposomes containing Ca2+-ATPase of sarcoplasmic reticulum and its hydrophobic fragment was investigated, using the method of synthetic penetrant ions and the radioisotopic method. The former method was used to determine the diffusional membrane potential formed by Ca2+ concentration gradient. It was demonstrated that Ca2+-ATPase, whose active center is oriented outside, has and asymmetric conductivity, i. e., it facilitates the rapid efflux of Ca2+ from proteoliposomes. This efflux is stimulated by the membrane potential positive inside. The hydrophobic fragment of Ca2+-ATPase forms a Ca2+-channel with a high conductivity for Ca2+. This channel is responsible for the Ca2+ efflux from sarcoplasmic reticulum.

[Passive Ca2+ fluxes across the membrane of sarcoplasmatic reticulum of skeletal muscles. The effect of calcium channel blockers]. / Issledovanie passivnykh potokov Ca2+ cherez membranu sarkoplazmaticheskogo retikuluma skeletnykh myshts. Vliianie blokatorov kal'tsievykh kanalov.

It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.

[Inhibition with tetracaine of passive Ca2+ flux through the membrane of the sarcoplasmic reticulum of skeletal muscles]. / Ingibirovanie tetrakainom passivnykh potokov Ca2+ cherez membranu sarkoplazmaticheskogo retikuluma skeletnykh myshts.

The initial fast phase of Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR) loaded with Ca2+ passively or in ATP-dependent process and the initial fast phase of Ca2+ influx into the SR vesicles are inhibited by 0.5 mM tetracaine. The inhibitory action of tetracaine observed both with the heavy and light SR fractions is competitive with Ca2+, decreased with varying pH from 5.8 to 7.8 and temperature from 20 degrees C to 37 degrees C. Choline chloride in the concentration of 150 mM at 37 degrees C prevents the inhibitory action of tetracaine. It is supposed that electrostatic interaction plays an essential role in tetracaine-inhibition of passive Ca2+ fluxes through the membrane.

[Effect of gradients of monovalent cations on active transport of Ca2+ in the sarcoplasmic reticulum and proteoliposomes]. / Vliianie gradientov odnovalentnykh kationov na aktivnyi transport Ca2+ v sarkoplazmaticheskom retikulume i proteoliposomakh.

Artificially generated K+ gradient from the sarcoplasmic reticulum vesicles enhances the ATP-dependent Ca2+ transport. The effect is not specific for K+, and is observed when K+ is replaced by Na+ or choline. Dissipation of the K+, Na+, choline gradient does not influence the ATP-dependent Ca2+ transport in proteoliposomes from asolectin and purified Ca2+-ATPase. The K gradient in the presence of valinomycin stimulates the ATP-dependent Ca2+ transport in proteoliposomes.

[Isolation and purification of proteolytic enzymes on organo-silica sorbents with immobilized gramicidin S]. / Vydelenie i ochistka proteoliticheskikh fermentov na organokremnezemnykh sorbentakh s immobilizirovannym gramitsidinom S.

Biospecific sorbents for affinity chromatography of proteolytic enzymes have been synthesized by attaching cyclopeptide antibiotic gramicidin S to organo-silica supports. It is shown possible to attach gramicidin S to the organo-silica supports using glutaric aldehyde, p-benzoquinone, soluble and insoluble carbodiimides. The sorbents prepared by these methods were successfully applied for the purification of the crude pepsin from horse gastric juice and proteolytic complex produced by Acremonium chrysogenum.

[Isolation of inosine-5'-monophosphate from fish muscles]. / Poluchenie inozin-5'-monofosfata iz myshts ryb.

Conditions for transformation of tissue adenosine-5'-monophosphate (AMP) into inosine-5'-monophosphate (IMP) with the aid of endogenic AMP-aminohydrolase are developed resting on the studied properties of AMP-aminohydrolase (EC 3.5.4.6) from saltwater fish muscles (one of the enzymes participating in the nucleotide metabolism). Sorption of the nucleotide is performed on the activated charcoals A gamma-3 A gamma-5 which eluate IMP from acid solutions. It reduces the process of isolation, permits application of the acid wash solutions to remove salts; the alkaline ethyl alcohol-aid elution at the subsequent stages accelerates the process of nucleotide concentration by means of vacuum evaporation. The suggested approaches allow developing a simple method of IMP production from fish tissues which diminishes the cost of preparation.

[Various properties of immobilized AMP-aminohydrolase]. / Nekotorye svoistva immobilizovannoi AMP-aminogidrolazy.

Properties of immobilized AMP-aminohydrolase from rabbit muscles are studied. The enzyme retains its activity for a year, is stable under manifold treatment with the substrate or under single treatment with 1 M NaCl which contains 50% ethylene glycol or 10% isopropanol and under treatment with 5 M K2 HPO4 (pH 8.5). The established pH-optimum (6.5-7.0) and the temperature optimum (30-40 degrees C) for immobilized AMP-aminohydrolase as well as inhibition of its activity by Co2+, Cd2+, Zn2+ and n-chloromercury benzoate indicate similarity of its properties with those of the purified enzyme.

[Changes in intravesicular pH during Ca2+ transport in the sarcoplasmic reticulum]. / Izmenenie vnutrivezikuliarnogo pH pri transporte Ca2+ v sarkoplazmaticheskom retikulume.

Studies with the use of [3H]acetate as an delta pH-indicator have established that pH in the native vesicles of sarcoplasmic reticulum is by 0.54 unit lower, than its extra-molecular value (6.5 units). The double [3H] and radioactive [3H] and [45Ca2+] labels were used to show that Ca2+ transport into the sarcoplasmic reticulum vesicles is accompanied by an increase in intravesicular pH. Carbonylcyanide-m-chlorophenylhydrazone, a protonophore, stimulates the equalization of the pH gradient (H+ removal) which is not accompanied by changes in the Ca2+ transport. In the presence of ionophore A23187 Ca2+ and [3H]acetate do not accumulate in vesicles in the ATP-dependent process. This indicates H+ removal from the vesicles only when there is the Ca2+ gradient creation and the absence of the close conjugation of Ca3+/2H+ realized by Ca2+-ATPase of sarcoplasmic reticulum.

[Changes in the intensity of the fluorescence of potential-sensitive fluorescent probes in the active transport of Ca2+ in the fragmented sarcoplasmic reticulum]. / Izmenenie intensivnosti fluorestsentsii potentsialchuvstvitel'nykh fluorestsentnykh zondov pri aktivnom transporte Ca2+ v fragmentirovannom sarkoplazmaticheskom retikulume.

The dependence of fluorescence intensity changes of potential-sensitive fluorescent probes 3,3'-dipropyl-2,2'-thyodicarbocianine and 1-anilino-8-naphtalenesulphonae on the ATP concentration during Ca2+ transport in fragmented SR of the rabbit skeletal muscle has been studied. An increase in the accumulation of Ca2+ in the SR vesicles caused by ATP is accompanied by an increase in the fluorescence intensity of the potential-sensitive probes. These fluorescence changes are related neither to ATP or Ca2+ effect but are coupled with cation accumulation inside the vesicles since they are not observed in the presence of either EGTA or triton X-100 or in the absence of Mg2+. The results obtained prove the membrane potential generation in SR in the course of ATP-dependent Ca2+ transport.

[Membrane potential during Ca++ transport and AMP deamination in vesicles of the sarcoplasmic reticulum]. / Membrannyi potentsial pri transporte Ca++ i dezaminirovanii AMP v vezikulakh sarkoplazmaticheskogo retikuluma.

Transport of Ca2+ to vesicles of the sarcoplasmic reticulum and its subsequent removal by AMP is accompanied by fluorescence intensification and quenching, respectively, of the charged potentially-dependent probes of 1-anilino-8-naphthalenesulphonate and 3,3'-dipropilthiodicarbocyanin as well as 4-dimethylaminochalcone responding to conformation changes in membranes. The transmembrane electrophoretic distribution of synthetic fat-soluble ions of phenyldicarboundecaborane and tetraphenylphosphonium during Ca2+ accumulation is characterized by uptake of phenyldicarboundecaborane with the subsequent removal by protonophore of m-cholorocarbonylcyanide phenylhydrazone or AMP; the uptake of tetraphenylphosphonium is absent. This testifies to the fact that the Ca2+ transport in vesicles is accompanied by the formation of a membrane potential with a positive sign inside the vesicles.

[Effect of ammonium ions on calcium release from fragmented sarcoplasmic reticulum]. / Vliianie ionov ammoniia na vykhod kal'tsiia iz fragmentirovannogo sarkoplazmaticheskogo retikuluma.

It is established that in AMP deamination by sarcoplasmic reticulum fragments there occurs an intensified release of the previously accumulated calcium. UMP has no noticeable effect on this process. The level of accumulated 45Ca+ in the fragmented sarcoplasmic reticulum is decreased when ammonium ions load is introduced into the medium. If the sarcoplasmic reticulum fragments were loaded with 45Ca2+ and then washed off and incubated in the isotonic sucrose solution, the calcium release is more intensified when ammonium ions are introduced into the medium. The results of ultrasound and A23187 treatment of the membranes evidence for the calcium release from the inner space of vesicles.