Allosteric regulation of cardiac sarcoplasmic reticulum Ca-ATPase: a comparative study.
The mechanisms of allosteric regulation of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum by ATP have been compared. Although both enzymes showed stimulation of ATPase activity by ATP, the cardiac enzyme did not show the plateau in ATPase activity at 10-100 microM ATP seen with the skeletal enzyme. Likewise the phosphoenzyme (EP) levels did not plateau with the cardiac enzyme as they did with the skeletal enzyme. The apparent negative cooperatively which was seen in the kinetics of ATP hydrolysis at low ATP concentrations was not due to negative cooperatively in substrate binding to either enzyme. The cardiac enzyme did show, however, much higher affinity for the ATP analog, AMPPCP, which helps explain how AMPPCP blocks ATPase activity in the cardiac enzyme and stimulates ATPase activity in the skeletal enzyme. Fluorescein isothiocyanate was used to determine if allosteric regulation takes place through site-site interactions in oligomers. The 1 to 1 ratio between AMPPCP binding sites and FITC binding sites eliminated allosteric regulation by effector sites in both enzymes. The allosteric mechanism which remained was one in which the active-site becomes an effector-site by the early departure of ADP in the reaction mechanism. The step stimulated by the binding of ATP at the active-site turned effector-site was a nonphosphorylated form of the enzyme in cardiac sarcoplasmic reticulum and a phosphorylated form in skeletal sarcoplasmic reticulum.
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Department of Physiology, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298.
Briggs
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FN
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013861:Thiocyanates / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate