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Isolation and phosphorylation of guinea pig cardiac sarcolemma Hui, Chi Wai

Abstract

Plasma membranes were prepared from guinea pig heart by homogenizing the tissue in a Polytron homogenizer, followed by four cycles of washing and differential centrifugation using low g forces (380 x g/10 minutes to 120 x g/10 minutes). The crude particles thus obtained were extracted with KCl (1.25 M), followed by isopycnic centrifugation in a discontinuous sucrose gradient. Adenylate cyclase was purified 10-15 fold over the whole homogenate with a specific activity of 3.6±0.72 nmoles/mg/minute. Ouabain-sensitive Mg⁺⁺-dependent Na⁺+K⁺-ATPase, another plasma membrane specific enzyme, was enriched by 4 fold, with a specific activity of 107±8.2 nmoles/mg/minute. Cytochrome C oxidase, an enzyme predominantly of mitochondrial origin, was recovered in low yield. These membrane "marker" enzyme studies indicated that the isolated membrane preparation consisted of highly purified plasma membranes. Functional studies with the cardiac sarcolemma indicated that an ATP-dependent Ca⁺⁺ binding system as well as Ca⁺⁺-dependent ATPases were present. Intrinsic protein kinase activities and membrane-bound substrates for phosphorylation were also found to be associated with these membranes, which were phosphorylated by endogenous or added protein kinase. Membrane phosphorylation was stimulated by cyclic AMP (1 μM) and was reversed by a membrane-bound phosphoprotein phosphatase, indicating the presence of a phosphorylation-dephosphorylation system in cardiac sarcolemma. Plasma membranes, when phosphor-ylated, were capable of accumulating twice as much Ca⁺⁺ as control preparations. For each nanomole of phosphate incorporated into the membrane, there were 15 nanomoles of net increase in Ca⁺⁺ uptake. These data are consistent with the possibility that cyclic AMP may facilitate Ca⁺⁺ movement into the cardiac cell via a phosphorylation-dephosphorylation mechanism.

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