Phosphorylation of low-molecular-weight proteins in preparations of rat heart sarcolemma and sarcoplasmic reticulum.
Two substrate proteins for cAMP-dependent protein kinase detected in a rat heart sarcolemma preparation displayed molecular weights of 24,000 and 9000 in sodium dodecyl sulfate gels and were shown to be interconvertible. The 9000-dalton protein could readily be separated from other low molecular weight phosphoproteins (mol. wt. 14,000 and 7000) by the use of 15% polyacrylamide gels. In addition to an endogenous cAMP-dependent protein kinase the membrane preparation also contained a protein-phosphorylation system that required Ca2+ and calmodulin. It appeared that both 24,000- and 55,000-dalton proteins were substrates for the endogenous Ca2+- and calmodulin-dependent protein kinase. Contaminating sarcoplasmic reticulum vesicles, first loaded with calcium oxalate, could be separated from the enriched sarcolemma preparation by sucrose gradient centrifugation. The separation was confirmed by comparative analysis of 5'-nucleotidase, Na+ -Ca2+ antiporter, and (Ca2+ + Mg2+)-dependent ATPase activities and by determination of gel electrophoretic (phospho)protein composition, sialic acid, cholesterol, and phospholipid contents. The 24,000-dalton phosphoprotein complex was equally distributed between sarcolemmal and sarcoplasmic reticulum fractions, whereas the 55,000- and 7000-dalton proteins were predominantly found in the sarcolemmal fraction. The 24,000-dalton protein was most likely phospholamban, because no other phosphoprotein was found in the 20,000 molecular weight range.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012508:Sarcolemma / Q000201:enzymology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D002147:Calmodulin; D000242:Cyclic AMP; D011494:Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium