Controlled proteolysis of the purified Ca2+-ATPase of the erythrocyte membrane. A correlation between the structure and the function of the enzyme.
The purified Ca2+-pumping ATPase of the erythrocyte plasma membrane has been subjected to a controlled proteolytic treatment with trypsin. The treatment has been previously shown to shift the enzyme from low to high Ca2+ affinity in the absence of calmodulin. The treatment leads to the fragmentation of the ATPase molecule into a number of products and to the accumulation of major limit polypeptides having Mr of 14,000, 28,000, 33,500, 48,000, and 76,000. The 33,500 Mr fragment reacts with 3(trifluoromethyl)-3 (m-[125I]iodophenyl)-diazirine, suggesting that it contains intramembrane regions of the enzyme. A number of minor fragmentation products were also formed, among them a polypeptide of Mr = 90,000 which is rapidly degraded further and a limit polypeptide of Mr about 25,000. [125I]Iodoazidocalmodulin cross-linked only to the original ATPase and to the fragments with Mr = 90,000 and Mr about 25,000. The Mr = 90,000 fragment can be isolated on calmodulin-Sepharose and on phenothiazine affinity columns. It retains the functional properties of the intact pump; it has a calmodulin-responsive ATPase activity and it actively accumulates Ca2+ upon incorporation into liposomes. Further proteolysis of the Mr = 90,000 fragment produces a fragment of Mr = 81,000-76,000 which forms an acylphosphate intermediate, which does not bind to calmodulin-Sepharose, and which seems to be responsible for the calmodulin-insensitive activity which appears as it is produced. A model is presented which postulates a major and a minor degradation pattern and rationalizes the pattern of proteolysis and of the [125I]iodoazidocalmodulin binding observed.
D001389:Azirines / Q000378:metabolism; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D001389:Azirines; D002147:Calmodulin; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D014357:Trypsin; D000252:Calcium-Transporting ATPases