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ATP PKODUCEU BY SARCOLEMMAL-BOUND CREATINE KINASE IS NOT PREFERENTIALLY USEU BY THE SUDIUM PUMP. Kenneth D. Philipson. Uepts. of Medicine and Physiology and the Cardiovascular Kesearch Laboratories, UCLA School of Medicine, Los Angeles, California, USA. It has been proposed that creatine kinase (CK) bound to cardiac sarcolemma (SL) can supply ATP directly to the Na pump (Grosse et al., BBA 603:142, 1980). We have investigated this hypothesis using cardiac SL vesicles and asensitive new technique for quantitating ATP-dependent Na transport. Na is actively pumped into inside-out vesicles and the intravesicular Na is quantitated by a subsequent Nai-dependent 4SCa uptake reaction (Na-Ca exchange). We find that added creatine phosphate (CP) stimulates Na pumping only under conditions when exogenously added ATP becomes depleted; i.e., maximal Na transport can occur if the added ATP supply is sufficient. ATP regenerated by exogenous pyruvate kinase/phosphoenolpyruvate or by endogenous CK/CP is used with equal efficiency by the Na pump. Na pumping supported by CK/CP is blocked by added hexokinase/glucose; this demonstrates that ATP generated by CK/CP enters the medium and is not delivered directly to the Na pump. Supported by NHLBI (HL-27821) and the Castera Foundation.
33O~&,~C~I7[~ II;:IIl;l'Ji(j;,, JE :,aL:;i:T S]~l{C3L,,~.~L /;.a+i'J-LfPase J'i p-JR();;~Pii~i~SiSjI'III3CY~l,',:l'~ /pJPITC/. A.Zie&elhdffer, A.&eier, A.Diurba. Institute of Xxperinenial Surgery, Center of Physiological Sciences. Slovak Academy of Sciences, iJ+2 33 Arrtislava, Czechoslovakia. Isothiocyanates as potent selective modifiers of thiol groups have been successfully applied in studying the active site structure of /Ja+K/-ATPose from the kidney. The aim of the present study was investigation of the mode of action and inhibitory effect of p31ITC on BDase activities of isolated rat lleart sarcolemmal /SL/ prcpara4~~~,d~~sl~"3-,"~4b.~~~~~-~~:~-Cs/3TPase activity. pYPITC.in concentraselectively and non-competitivre+y innlted the /Ma+ij/-ATPase activity in SL jvith an ID ; around 2.1'32+ mol.1 , Xowever, any inhibitory effect was observed 5deither on i9g -i'.TPase cind.in contrery to those of the sarcoplesrtic rotictulum nor on the CaCf -A.pPa-e u activity of SL. The non-2g-;ecific interaction of pJITC with bivrlent cations namely with I& in the reaction system was eliminated by preincubetion-of membranes xith p.WITC keeping the ratio of inhibitor tQ rne:;lbrane protein constant. lreincuaation of membranes with 2 mr.101.1 -I BTP eliminated the inhibitory action of gd?ITC. The interaction of inhibitor with the SL was reversible in t e presence of beta-merkaptoethanol or dithiothreitol.
361THE INVOLVEMENT +Mg2+)ATPase. (Cd+ Medical Faculty,
OF CALMODULIN IN J.M.J. Lamers Erasmus University
THE REGULATION and J.T. Stinis. Rotterdam,
OF SARCOLEMMAL Department The Netherlands.
of
PROTEIN KINASE Biochemistry
AND I,
dependent protein Cardiac sarcolemmal vesicles (SL) contain a Ca 2+-calmodulin kinase (CaM-PK), which most important substrate proteins have molecular weights of 55 and 9 kD (pig, dog and rat), 40, 43 and 9 kD (rabbit). The CaM-PK reached halfmaximal rate at 1 UM Ca2+ III the presence of 0.5 PM CaM. The affinity for CaM was 60 nM at 12 llM Ca*+. The anti CaM drug calmidazolium decreased the maximal activity and its inhibition could be reversed by increasing CaM. The (Ca*++Mg*+)ATPase, which was inhibited by the anti CaM drug in was also present in this membrane preparation, a different manner: it decreased the apparent affinity of the ATPase for Ca2+ ions. Exogenous CaM was again able to antagonize drug inhibition, however, the ATPase activity was not dependent on exogenous CaM. Membrane-bound CaM, supposed to be Involved in the regulation of Ca2+ sensitivity of the (Ca*++Mg*+)ATPase, was estimated with a &M-deficient phosphodiesterase. It appeared to be present in 1:2 stoichiometry compared to the phosphoenzyme intermediate, which was estimated by Ca2+-dependent " P incorporation on SDS-PAGE. Supported by the Dutch Heart Foundation.
ATP PKODUCEU BY SARCOLEMMAL-BOUND CREATINE KINASE IS NOT PREFERENTIALLY USEU BY THE SUDIUM PUMP. Kenneth D. Philipson. Uepts. of Medicine and Physiology and the Cardiovascular Kesearch Laboratories, UCLA School of Medicine, Los Angeles, California, USA. It has been proposed that creatine kinase (CK) bound to cardiac sarcolemma (SL) can supply ATP directly to the Na pump (Grosse et al., BBA 603:142, 1980). We have investigated this hypothesis using cardiac SL vesicles and asensitive new technique for quantitating ATP-dependent Na transport. Na is actively pumped into inside-out vesicles and the intravesicular Na is quantitated by a subsequent Nai-dependent 4SCa uptake reaction (Na-Ca exchange). We find that added creatine phosphate (CP) stimulates Na pumping only under conditions when exogenously added ATP becomes depleted; i.e., maximal Na transport can occur if the added ATP supply is sufficient. ATP regenerated by exogenous pyruvate kinase/phosphoenolpyruvate or by endogenous CK/CP is used with equal efficiency by the Na pump. Na pumping supported by CK/CP is blocked by added hexokinase/glucose; this demonstrates that ATP generated by CK/CP enters the medium and is not delivered directly to the Na pump. Supported by NHLBI (HL-27821) and the Castera Foundation.
33O~&,~C~I7[~ II;:IIl;l'Ji(j;,, JE :,aL:;i:T S]~l{C3L,,~.~L /;.a+i'J-LfPase J'i p-JR();;~Pii~i~SiSjI'III3CY~l,',:l'~ /pJPITC/. A.Zie&elhdffer, A.&eier, A.Diurba. Institute of Xxperinenial Surgery, Center of Physiological Sciences. Slovak Academy of Sciences, iJ+2 33 Arrtislava, Czechoslovakia. Isothiocyanates as potent selective modifiers of thiol groups have been successfully applied in studying the active site structure of /Ja+K/-ATPose from the kidney. The aim of the present study was investigation of the mode of action and inhibitory effect of p31ITC on BDase activities of isolated rat lleart sarcolemmal /SL/ prcpara4~~~,d~~sl~"3-,"~4b.~~~~~-~~:~-Cs/3TPase activity. pYPITC.in concentraselectively and non-competitivre+y innlted the /Ma+ij/-ATPase activity in SL jvith an ID ; around 2.1'32+ mol.1 , Xowever, any inhibitory effect was observed 5deither on i9g -i'.TPase cind.in contrery to those of the sarcoplesrtic rotictulum nor on the CaCf -A.pPa-e u activity of SL. The non-2g-;ecific interaction of pJITC with bivrlent cations namely with I& in the reaction system was eliminated by preincubetion-of membranes xith p.WITC keeping the ratio of inhibitor tQ rne:;lbrane protein constant. lreincuaation of membranes with 2 mr.101.1 -I BTP eliminated the inhibitory action of gd?ITC. The interaction of inhibitor with the SL was reversible in t e presence of beta-merkaptoethanol or dithiothreitol.
361THE INVOLVEMENT +Mg2+)ATPase. (Cd+ Medical Faculty,
OF CALMODULIN IN J.M.J. Lamers Erasmus University
THE REGULATION and J.T. Stinis. Rotterdam,
OF SARCOLEMMAL Department The Netherlands.
of
PROTEIN KINASE Biochemistry
AND I,
dependent protein Cardiac sarcolemmal vesicles (SL) contain a Ca 2+-calmodulin kinase (CaM-PK), which most important substrate proteins have molecular weights of 55 and 9 kD (pig, dog and rat), 40, 43 and 9 kD (rabbit). The CaM-PK reached halfmaximal rate at 1 UM Ca2+ III the presence of 0.5 PM CaM. The affinity for CaM was 60 nM at 12 llM Ca*+. The anti CaM drug calmidazolium decreased the maximal activity and its inhibition could be reversed by increasing CaM. The (Ca*++Mg*+)ATPase, which was inhibited by the anti CaM drug in was also present in this membrane preparation, a different manner: it decreased the apparent affinity of the ATPase for Ca2+ ions. Exogenous CaM was again able to antagonize drug inhibition, however, the ATPase activity was not dependent on exogenous CaM. Membrane-bound CaM, supposed to be Involved in the regulation of Ca2+ sensitivity of the (Ca*++Mg*+)ATPase, was estimated with a &M-deficient phosphodiesterase. It appeared to be present in 1:2 stoichiometry compared to the phosphoenzyme intermediate, which was estimated by Ca2+-dependent " P incorporation on SDS-PAGE. Supported by the Dutch Heart Foundation.