Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody.
Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.
Ogurusu
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Wakabayashi
S
S
Furukawa
K
K
Tawada-Iwata
Y
Y
Imagawa
T
T
Shigekawa
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D008168:Lung / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D012508:Sarcolemma / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases; D002118:Calcium