Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Abstract

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na + + K +)-ATPase activity of 28.0 ± 1.5 μmol P i/mg protein per h and the high concentration of muscarinic receptors with the B max of 8.2 ± 2.5 pmol/mg protein as determined by [ 3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca 2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol P i/mg per min, in the presence of 6.25 μg phosphatidylserine and 0.5 mM CaCl 2. Treatment of sarcolemma with 12- O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu 2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu 2 on protein kinase C in sarcolemma was independent of exogenous Ca 2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol P i/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol P i/min, compared to that of 1025 pmol P i/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca 2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of M r 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of M r 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca 2+ only. Phosphorylation of phospholamban ( M r 27 000 and 11 000) was also stimulated in the presence of Ca 2+ and phosphatidylserine, but the low M r form of phospholamban was distinct from two other low M r substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and an M r 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.