Human leukemia CEM C-1 cells possess a high affinity binding site for farnesol.

Binding of 15-carbon isoprenoid farnesol in human acute leukemia CEM C-1 cells has been studied by addition of radio-labeled isoprenoid to cell growth medium. Significant time-dependent accumulation of the cell-associated radioactivity was detected at 37 degrees C. When experiments were carried out at 4 degrees C, about 10 times decrease in cell labeling was observed. In contrast, binding of farnesol by the liposomes prepared from cellular lipids was independent of the temperature. When binding experiments were performed in the presence of the excess of unlabeled farnesol, a saturable specific component was separated from the total binding. Analysis of the specific binding revealed about 335,000 binding sites per cell with Kd of 5.9 x 10(-8) M. The data suggest that CEM C-1 cells may possess receptors capable of binding farnesol.

[Ca2+ metabolism in the sarcoplasmic reticulum of skeletal muscle after surgical denervation]. / Obmen Ca2+ v sarkoplazmaticheskom retikulume skeletnykh myshts pri khirurgicheskoi denervatsii.

The ability of sarcoplasmic reticulum to regulate Ca(2+)-metabolism was studied at different terms after surgical denervation. The increase of Ca(2+)-ATP-ase activity, intensification of active Ca(2+)-accumulation under insignificant change of passive Ca(2+)-outflow from sarcoplasmic reticulum vesicles were observed 3-days after denervation. At more remote terms 14 days and 28 days after denervation all these parameters decreased except for Km: Ca(2+)-ATP activity and active Ca2+ accumulation decreased below normal level.

[Effect of fluoroaluminate on the passive transport of calcium in the plasma membrane fraction of the pig myometrium]. / Vliianie ftoroaliuminata na passivnyi transport kal'tsiia vo fraktsii plazmatickeskikh membran miometriia svin'i.

Study of the effect of aluminium, fluoride-ions and fluoroaluminium on the passive transport of Ca2+ through the plasma membrane of the myometrium cells has shown that 10 microM of Al3+, 1 mM of F- and the both ions taken in the mentioned concentrations do not affect this process. At the same time 10 mM of F- as well as 10 microM of Al3+ together with 10 mM of F- inhibit a passive output of calcium from vesicles. The results obtained suppose that inhibitory regulation of the passive transport of calcium in the myometrium sarcolemma is executed through Gi-protein.

[Inhibiting effect of ADP-ribosylation by pertussis toxin on passive transport of Ca2+ into the cell membrane of porcine myometrium]. / Ingibiruiushchee deistvie ADP-ribozilirovaniia kokliushnym toksinom napassivnyi transport Ca2+ v plazmaticheskoi membrane miometriia svin'i.

ADP-ribosylation by whooping cough toxin of protein components of inside-out oriented vesicles of pig myometrium plasma membranes under conditions of their depolarization results in significant inhibition of passive transport of Ca2+ ions. The inhibiting effect is dose- and time-dependent. rho-Chloromercuribenzoate (0.5 mM) blocks the effect of whooping cough toxin, no such effect on Ca2+ transport being observed in control preparations.

[The effect of antibodies against Ca2(+)-ATPase and its hydrophobic fragment on the activity of this enzyme and on the passive transport of Ca2(+)]. / Vliianie antitel protiv Ca2(+)-ATPazy i ee gidrofobnogo fragmenta na aktivnost' étogo fermenta i passivnyi transport Ca2(+).

It is shown that both Ca(2+)-ATPase and its hydrophobic fragment are immunogenic factors. A region between hydrophobic and hydrophilic parts of Ca(2+)-ATP-ase is immunogenic. These antibodies clearly inhibit inflow and outflow of Ca2+ through a hydrophobic fragment reconstructed into liposomes and do not influence Ca(2+)-ATPase activity.

[Analysis of the role of calcium in analgesic action of non-narcotic analgesics and calcium channel blockers]. / Analiz roli kal'tsiia v obezbolivaiushchem deistvii nenarkoticheskikh anal'getikov i blokatorov kal'tsievykh kanalov.

In the mice hot-plate test we have compared analgesic effect of calcium channel blockers and new non-narcotic analgesic antiinflammatory agent PV-107: verapamil > fenigidin > PV-107. Simultaneously we have shown strong correlation (r - 0.82) between analgesic effect and 45Ca2+ efflux of cardiac membrane in depolarizing media in vitro.

[Phosphorylation of myocardial sarcolemma proteins during induction of the phosphatidylinositol cycle in isolated perfused rabbit heart]. / Fosforilirovanie belkov sarkolemmy miokarda pri induktsii fosfatidilinozitidnogo tsikla v izolirovannom perfuziruemom serdtse krolika.

Phosphorylation of cardiac sarcolemma proteins under stimulation of M-receptors by agonist carbacholine used to stimulate phosphatidylinositide cycle, was investigated in the isolated, rabbit heart perfused with 32Pi. Carbacholine (10(-7) stimulates the polyphosphoinositide metabolism which is expressed in the activated incorporation of 32P from [gamma-32P]ATP in polyphosphoinositide as well as in the increased content of the labelled inositol trisphosphate released through phosphatidylinositol-4,5-bisphosphate break-down by phospholipase C. The diacylglycerol produced simultaneously with inositol triphosphate as a second messenger activates the protein kinase C. This was confirmed by considerable activation of phosphorylation sarcolemma proteins-substrates of protein kinase C, with Mr 94, 87, 78, 51 and 46 kDa.

The ion-conducting properties of Ca2+ ATPase in rabbit skeletal muscle sarcoplasmic reticulum.

Ca2+ ATPase was isolated from rabbit skeletal muscle sarcoplasmic reticulum and used to form structures resembling potential-dependent calcium channels within the membrane lipid bilayer of liposomes. The orientation of these structures in the bilayer was dependent on the conditions used for enzyme incorporation. The results obtained indicate that Ca2+ ATPase may be involved in the passive transport of calcium ions from the sarcoplasmic reticulum which may be regulated by the membrane potential. The membrane potential within the reticulum is probably positive at the moment of calcium ion release.

[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.

[Catalytic properties of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma]. / Kataliticheskie svoistva ochishchennoi Ca2+,Mg2+-ATPazy sarkolemmy miometriia.

The catalytic properties of myometrium sarcolemmal Ca2+, Mg2(+)-ATPase purified from plasma membrane solubilizate by affinity chromatography on calmodulin-Sepharose were investigated. The enzyme isolated in the presence of azolectin revealed a calmodulin-independent affinity for Ca2+ (Km = 0.17 microM). Purified Ca2+, Mg2(+)-ATPase displayed a strict substrate specificity, was inhibited by low concentrations of o-vanadate and was insensitive to oxytocin and prostaglandins E2 and F2 alpha. The enzyme activity was maximal at 45 degrees C, pH 7.5-8.0, and at Mg-ATP and Ca2+ concentrations of 1.5-2.5 mM and 5-20 microM, respectively.

[Calmodulin-induced activation of ATP-dependent Ca2+ transport in plasma membranes of the myometrium]. / Aktivatsiia kal'modulinom ATP-zavisimogo transporta Ca2+ v plazmaticheskikh membranakh miometriia.

Calmodulin activates the ATP-dependent transport of Ca2+. The V0 value for this reaction in the absence of calmodulin is 0.82, that in the presence of 10(-7) M calmodulin is 5 times as high, i. e. 4.5 nmol 45Ca2+/mg protein/min. The Vmax value in the absence of calmodulin is 2.07, that with the activator is 4.33 nmol 45Ca2+/mg protein/min. The corresponding Km values are 0.75 X 10(-6) M and 0.66 X 10(-7) M, respectively, i. e., the affinity of the Ca-pump for Ca2+ increases. The half-maximum Ca-binding activity of calmodulin measured with a help of the fluorescent probe, N-phenyl-1-naphthylamine (PNA), is observed at 5 X 10(-7) M Ca2+. Mg2+ (3 mM) decreases 10-fold the Ca-binding affinity. No significant effect of ATP on the Ca-binding properties of calmodulin was found; the Hill coefficient is suggestive of a positive cooperativity of this reaction. A comparison of dependences of the calmodulin-stimulated component of ATP-dependent transport of Ca2+ in myometrium plasma membranes and of the Ca-binding activity of calmodulin measured with a help of PNA suggests that the effect of calmodulin on the affinity of the Ca-pump for Ca2+ can also be realized when some (but not all) Ca-binding sites in the calmodulin molecule are saturated with Ca2+.

[Reconstruction of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma into liposomes and its catalytic properties]. / Rekonstruktsiia ochishchennoi Ca2+,Mg2+-ATP-azy sarkolemmy miometriia liposomy i ee kataliticheskie svoistva.

The Ca2+, Mg2(+)-ATPase of the myometrium sarcolemma purified by the method of affinity chromatography on calmodulin sepharose is reconstituted into azolectin liposomes in the functionally active form by means of cholate dialysis. The ATPase-dependent accumulation of 45Ca is shown on the obtained model system. It makes up 95% of the total accumulation and may decrease to 43% under the effect of 0.8 microM A23187. Ca2+, Mg2(+)-ATPase reconstituted into azolectin liposomes is in the high affinity to Ca2+; Km for Ca2+ is equal to 0.88 +/- 0.22 microM, calmodulin practically does not change it. The highest activity of the reconstituted enzyme is observed at pH 7.0, temperature 50 degrees C, the Mg-ATP concentration 1-2 mM. The Km for substrate is 0.45 +/- 0.02 mM.

[Isolation and purification of Ca2+,Mg2+-ATPase from plasma membranes of the myometrium]. / Vydelenie i ochistka Ca2+,Mg2+-ATP-azy plazmaticheskikh membran miometriia.

The preparation of the purified Ca2+, Mg2(+)-ATPase has been isolated from triton X-100 solubilizate of plasma membranes of the pig myometrium using the method of affinity chromatography on calmodulin-Sepharose 4B. The specific activity of the enzyme shows its 52-fold purification. The enzymic preparation practically has no Mg2(+)-ATPase activity. By the data of DS-Na-electrophoresis in PAAG the Ca2+, Mg2+ ATPase preparation consists of two polypeptides with Mm 130 and 205 kDa. Autoradiography shows their Ca2(+)-dependent phosphorylation. The purified enzyme is highly sensitive to the inhibitory effect of orthovanadate.

[Calcium transport in endoplasmic reticulum of the rat liver during lipid peroxidation]. / Transport kal'tsiia v éndoplazmaticheskom retikulume pecheni krys pri aktivatsii perekisnogo okisleniia lipidov.

Some parameters of calcium transport in rat liver microsomes under conditions of lipoperoxidation activation modelled by antioxidant deficiency (AOD) were studied. This process was shown to be associated with a sharp stimulation of NADPH- and ascorbate-dependent lipid peroxidation in hepatocyte endoplasmic reticulum. The activation of lipid peroxidation was accompanied by disturbances in the kinetic properties of Ca2(+)-ATPase. This was paralleled with a considerable decrease of the ATP-dependent 45Ca-accumulation, increase in the passive permeability of microsomal vesicles for Ca2+ and Ca2+ elevation in the microsomal fraction. The AOD-induced diminution of the Ca2(+)-pump efficiency was slightly prevented by injections of rats with the antioxidants, alpha-tocopherol acetate and ionol which enable Ca2+ compartmentation correction in liver cytosol and membrane fractions.

[The effect of ionizing radiation on the structure of the hydrophobic fragment of Ca-ATPase in skeletal muscle sarcoplasmic reticulum]. / Vliianie ioniziruiushchei radiatsii na strukturu gidrofobnogo fragmenta Ca-ATFazy sarkoplazmaticheskogo retikuluma skeletnykh myshts.

Early (1 and 24 h) after X-irradiation with a dose of 0.21 C/kg changes occurred in the acceptability of the polypeptide chain parts of sarcoplasmic reticulum Ca-ATPase for the effect of trypsin. The analysis of the results of studying the structural and functional properties of a hydrophobic fragment of this enzyme in the control and after irradiation permitted to define the part of the Ca-ATPase polypeptide chain that provided ion selectivity of the fragment.

[A mechanism of passive transport of Ca2+ in muscle sarcoplasmic reticulum]. / Mekhanizm passivnogo transporta Ca2+ v sarkoplazmaticheskom retikulume myshts.

Basing on the data available in literature and authors' investigations the mechanism of local alkalization of the myoplasm by proton efflux attended by Ca2+ influx is mic reticulum and may be the main link in the process of electrochemical coupling in the skeletal and cardiac muscle cells. Experimental evidence for participation of Ca2(+)-ATPase in the passive transport of calcium through sarcoplasmic membrane is given.

[The effect of Ca2+-phospholipid dependent phosphorylation on passive calcium transport in the myocardial sarcolemma]. / Vliianie Ca2+-fosfolipidzavisimogo fosforilirovaniia na passivnyi transport kal'tsiia v sarkolemme miokarda.

Protein kinase C in vesicular preparations of the myocardium sarcolemma is shown to phosphorylate proteins with the molecular weight of 250, 140, 67, 58, 24 and 11 kD. The exogenic protein kinase C catalyzed phosphorylation of the sarcolemma preparations lowers the initial rate of the passive calcium transport from 0.56 down to 0.18 mmol per 1 mg second. Activation of endogenic protein kinase C by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate is also accompanied by phosphorylation of vesicular preparations of sarcolemma and by inhibition of the passive calcium transport. Polymyxin B, being an inhibitor of protein kinase C, suppresses the phosphorylation and thus prevents the inhibitory action of phosphorylation on the passive calcium transport.

[ATP-dependent transport of calcium in the myocardial sarcoplasmic reticulum during adaptation to muscular activities]. / ATP-zavisimyi transport kal'tsiia v sarkoplazmaticheskom retikulume miokarda pri adaptatsii k myshechnoi deiatel'nosti.

Two-fold increase in Ca2+-transport properties of Ca2+-pump was detected in myocardial sarcoplasmic reticulum (SR) during adaptation of animals to muscular activity (training by means of running on treadmill within 6 weeks, with gradual increase in duration and intensity). Besides, the rate of SR membranes phosphorylation via cAMP-dependent protein kinase was increased 1.3-fold. At the same time, cAMP-dependent phosphorylation accelerated 1.4-1.5-fold the rate of ATP-dependent transport of Ca2+ in myocardial SR of both control and trained animals. The data obtained suggest that increase in the Ca2+-pump activity and in the rate of phosphorylation of SR membranes via cAMP-dependent protein kinase are of importance for adaptation of animal heart muscle to elevated activity.

[Ca2+ (calmodulin)-dependent phosphorylation of plasma membranes of pig myometrium]. / Ca2+(Kal'modulin)-zavisimoe fosforilirovanie plazmaticheskikh memran miometriia svin'i.

The high-purified vesicles of pig myometrium sarcolemma closed, mainly, so that the cytoplasmatic side is outside possess the Ca2+ (calmodulin)-dependent protein kinase activity. The initial rate of the endogenic phosphorylation without exogenic calmodulin is 6.3 and with its presence--10.7 pmol of 32Pi 1 min per 1 mg of protein. Km for ATP is equal to 164 microM, and Vmax--0.27 nmol of 32Pi 1 min per 1 mg of protein. Exogenic calmodulin increases the affinity to ATP (50 microM), Vmax being unchanged. Under optimal concentrations of calmodulin (10(-7)-10(-6) M) and 10(-4) M Ca2+ the protein kinase activity is 0.132 nmol of 32Pi min per 1 mg of protein. Electrophoresis in DS-PAAG has shown that membrane proteins with molecular weight of 105, 58, 25, 12 and 2 kDa are basic substrates of Ca2+ (calmodulin)-dependent phosphorylation. Trifluoperazine++ in the concentration of 40 microM inhibits phosphorylation of all five proteins. Ca2+ (calmodulin)-dependent phosphorylation is supposed to be a regulator of Ca2+-transport processes of sarcolemma.