Chick heart plasma membranes. Isolation and analysis of autophosphorylation.
Plasma membranes have been isolated from hearts of 10-day embryonic and newborn chicks. The membranes obtained were highly enriched in muscarinic acetylcholine receptors, K+ -stimulated, ouabain-sensitive p-nitrophenylphosphatase and 5'-nucleotidase. There was little contamination of the membrane fractions by the mitochondrial membranes or by contractile proteins. The autophosphorylation of the isolated membrane fractions was analyzed by measuring 32P incorporation from [gamma-32P]ATP into total membrane protein and into individual membrane components. Membranes obtained from embryonic hearts contained significantly more cAMP-dependent and -independent protein kinase activities than membranes from newborn chick hearts. Treatment of the membranes with Triton X-100 or the peptide ionophore alamethicin increased phosphorylation in membranes from either newborn or embryonic hearts. Membranes from embryonic hearts contained substrates for membrane-bound cAMP-dependent and -independent protein kinases either not observed or present in low amount in membranes from newborn hearts, and vice-versa. Notably, a 38 kDa protein was markedly phosphorylated by endogenous cAMP dependent protein kinase in plasma membrane enriched fractions from embryonic hearts. This phosphoprotein was not easily detected in any fraction obtained from newborn hearts. One cAMP-dependent phosphoprotein had an Mr of 27000 or 11000, depending on the conditions used to solubilize it. This protein was present in sarcolemma-enriched membranes as well as membrane fractions containing sarcoplasmic reticulum. There was more of this phosphoprotein in newborn heart membranes than in embryonic hearts. The phosphorylation of this protein was markedly enhanced by the peptide ionophore alamethicin. A second cAMP-dependent phosphoprotein with an Mr of 27000 was also detected in the sarcolemma-enriched membranes.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D002642:Chick Embryo; D002645:Chickens; D008565:Membrane Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D008565:Membrane Proteins; D011494:Protein Kinases