Phosphorylation and dephosphorylation of purified phospholamban and associated phosphatidylinositides.
Phospholamban, the putative regulator for the calcium pump, was purified to apparent homogeneity and in high yields from canine cardiac sarcoplasmic reticulum membranes. Purified phospholamban migrated with an apparent Mr of 27,000 in alkaline sodium dodecyl sulfate-polyacrylamide gels, and upon boiling in 7.5% sodium dodecyl sulfate, it dissociated into a lower molecular weight component of 5500-6000. Purified phospholamban contained 0.62 +/- 0.09 mumol of lipid Pi/mg of protein, and the major phospholipids were phosphatidylserine (34%), phosphatidylcholine (22%), sphingomyelin (17%), phosphatidylinositol (13%), and phosphatidylethanolamine (9%). Phospholamban was phosphorylated by cAMP-dependent protein kinase to a level of 207 nmol of Pi/mg, and this would indicate an incorporation of 1 mol of phosphate/mol of protein, assuming a molecular weight of 5500 for phospholamban. Phosphorylation of phospholamban could be reversed by a "phospholamban phosphatase" isolated from canine cardiac cytosol. Phospholipids associated with the purified phospholamban were also phosphorylated in the presence of the catalytic subunit of cAMP-dependent protein kinase, and the maximal phosphate incorporation was 4 nmol/mg of protein. The main phospholipids phosphorylated were phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate. Phosphorylation of phospholipids was inhibited by the heat-stable inhibitor protein of the cAMP-dependent protein kinase, and it could be also reversed by the phospholamban phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
Jakab
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Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Kranias
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EG
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004285:Dogs; D005227:Fatty Acids / Q000032:analysis; D007700:Kinetics; D008563:Membrane Lipids / Q000032:analysis; D009206:Myocardium / Q000201:enzymology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D005227:Fatty Acids; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D010743:Phospholipids; D000099211:Phospholamban