Partial purification and characterization of thrombolamban, a 22,000 dalton cAMP-dependent protein kinase substrate in platelets.
In preparations of human platelet microsomes, cyclic AMP-dependent protein kinase induced the rapid phosphorylation of a single protein that was electrophoretically identical to the 22,000 dalton protein (P22) phosphorylated by cAMP in intact platelets. Phosphorylation of the microsomal protein was maximal at one minute and was followed by slow dephosphorylation. Although the protein was associated with a microsomal fraction, it could be separated from the membrane by 2 M NaCl indicating that it was a peripheral protein. Molecular weight was estimated by NaDodSO4-PAGE and by gel filtration chromatography. The molecular weight estimated by NaDodSO4-PAGE was 22,400 daltons and was somewhat larger than the 16,000 molecular weight estimated by gel filtration in the presence of NaDodSO4. In the absence of NaDodSO4, the protein chromatographed as a 36,000 dalton form. The presence of the 36,000 dalton form was not dependent on the phosphorylation state of the protein. The partially purified protein contained phosphoserine, but no phosphothreonine or phosphotyrosine. Two dimensional NaDodSO4-PAGE and isoelectric focusing of the phosphorylated protein revealed isomers with pl values of 5.9 and 6.3. These studies indicate that the 22 kDa microsomal protein and P22 in intact platelets are the same protein and that the 22 kDa protein is tightly bound to the microsomal membrane although the nature of this binding and the microsomal component(s) to which it is bound remain to be determined. We conclude that the 22 kDa protein in platelet microsomes is structurally distinct from, but functionally similar to, phospholamban, the cAMP-dependent protein kinase substrate in muscle, and may play a similar role in calcium transport. Based on this similarity, it is proposed that the 22 kDa protein in platelets be called thrombolamban.
Department of Medicine, University of North Carolina, Chapel Hill 27514.
T H
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Fischer
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White
D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000032:analysis*; D001798:Blood Proteins / Q000302:isolation & purification*; D002135:Calcium-Binding Proteins / Q000032:analysis; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification*; D010766:Phosphorylation; D011494:Protein Kinases; D012965:Sodium Chloride / Q000494:pharmacology; D000099211:Phospholamban
D000596:Amino Acids; D001798:Blood Proteins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D012965:Sodium Chloride; D000099211:Phospholamban; C054412:thrombolamban