The effect of β-adrenergic stimulation on sarcolemmal protein phosphorylation was examined in intact ventricular myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with 32P(i) after which membrane vesicles enriched 3-5 fold in sarcolemma were isolated by differential centrifugation followed by sucrose gradient centrifugation. Perfusion of hearts with isoproterenol stimulated 32P incorporation into a protein of apparent molecular weight of 15,000, which copurified with sarcolemmal vesicles. The increase in 32P incorporation was rapid in onset and elevated 2.5-3.0-fold after 30-45 s exposure of hearts to 100 nM isoproterenol. A positive correlation was found between stimulation of phosphorylation of the 15-kDa protein and the increase in the maximal rate of developed tension in intact ventricles after administration of isoproterenol. Phosphorylated phospholamban (most likely present as a contaminant) was also identified in the same sarcolemmal preparations. However, phospholamban and the 15-kDa sarcolemmal substrate were different proteins. Boiling of the membrane samples in sodium dodecyl sulfate prior to electrophoresis dissociated the high M(r) form of phospholamban into the form of lower M(r) but did not alter the mobility of the 15-kDa protein in sodium dodecyl sulfate-polyacrylamide gels. The 15-kDa protein did not undergo the electrophoretic mobility shift that is characteristic of phospholamban after cAMP-dependent phosphorylation nor did it cross-react with a highly specific phospholamban antibody. In vitro the unmasking agent Triton X-100 suggested that the 15-kDa protein was localized to the cytoplasmic surfaces of sarcolemmal vesicles. These results demonstrate phosphorylation of a sarcolemmal protein, distinct from phospholamban, in response to β-adrenergic stimulation of the heart. Phosphorylation of the sarcolemmal 15-kDa protein may play a role in mediating the effects of β-adrenergic agonists on cardiac contractile force.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1985|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology