Effect of cyclic AMP-dependent phosphorylation on the reactivity of sulphydryl groups in cardiac sarcoplasmic reticulum.
Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase results in enhanced Ca2+ transport even though Ca2+-dependent ATPase is not a substrate for the kinase. The mechanisms involved in this enhancement are not clear. In the present study, we used the reactivity of sulphydryl groups in the Ca2+-dependent ATPase as an index of conformational change during the Ca2+ transport cycle and examined the effects of protein kinase-catalysed phosphorylation. N-Ethylmaleimide alkylation allowed the distinction of several thiol groups with variable functional significance for the ATPase. A sulphydryl group involved in the formation of the phosphorylated intermediate (EP) of the Ca2+-dependent ATPase was protected by adenosine 5'-[beta, gamma-imido]triphosphate. Reactivity of an additional thiol group was related to EP dephosphorylation and was dependent on Ca2+. The Ca2+ concentration for change in the reactivity of this sulphydryl group and ATPase inhibition occurred within the range for Ca2+ binding to the high-affinity sites. Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase resulted in decreased N-ethyl[1-14C]-maleimide binding and the ATPase inhibition; the thiol groups involved in EP dephosphorylation were selectively protected. The results indicate that protein kinase-catalysed phosphorylation results in conformational changes of the ATPase, which renders certain thiol groups inaccessible to N-ethylmaleimide. This conformational change may facilitate functional movements involved in Ca2+ transport.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000378:metabolism; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008861:Microsomes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D000242:Cyclic AMP; D000252:Calcium-Transporting ATPases; D005033:Ethylmaleimide