cAMP-dependent protein kinase substrates in platelets. Evidence that thrombolamban, a 22,000 dalton substrate, and the Ca++-ATPase are not associated proteins.
Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase.
Fischer
T H
TH
Department of Medicine, University of North Carolina, Chapel Hill 27599.
White
G C
GC
D001792:Blood Platelets / Q000201:enzymology*; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D003432:Cross-Linking Reagents; D004347:Drug Interactions; D006801:Humans; D008861:Microsomes / Q000032:analysis; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity
D001798:Blood Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D010750:Phosphoproteins; C054412:thrombolamban; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases