Immunological detection of phospholamban phosphorylation states facilitates the description of the mechanism of phosphorylation and dephosphorylation.
Six electrophoretically distinct species of oligomeric phospholamban were identified immunologically following phosphorylation of sarcoplasmic reticulum vesicles by cAMP-dependent protein kinase. The phosphate content of each was determined, confirming that the discrete sequential retardation of phospholamban oligomers was the result of ascending mole ratios of phosphate (P0-P5) per oligomer. These data afford support to the pentameric arrangement of oligomeric phospholamban and offer a means of determining phosphorylation stoichiometry independent of the absolute phospholamban concentration. Detection of the relative concentration of individual species during phosphorylation facilitated the description of a random mechanism of phosphorylation by cAMP-dependent protein kinase. By contrast, dephosphorylation of cAMP-dependent protein kinase phosphorylated phospholamban was shown to exhibit strong positive cooperativity in its reaction mechanism.
Li
C F
CF
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Wang
J H
JH
Colyer
J
J
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D004285:Dogs; D007120:Immunochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D000099211:Phospholamban
D000255:Adenosine Triphosphate; D002135:Calcium-Binding Proteins; D011494:Protein Kinases; D000099211:Phospholamban; C022571:adenosine 5'-O-(3-thiotriphosphate)