Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis.
In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.
D000251:Adenosine Triphosphatases / Q000097:blood; D000626:Aminopeptidases / Q000378:metabolism; D018826:CD13 Antigens; D002118:Calcium / Q000097:blood* / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002918:Chymotrypsin / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007811:Lanthanum / Q000494:pharmacology; D010206:Papain / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010710:Phosphates / Q000097:blood; D011402:Pronase / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D007473:Ion Channels; D010710:Phosphates; D007811:Lanthanum; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D018826:CD13 Antigens; D043422:Carboxypeptidases A; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain; D011402:Pronase; D000251:Adenosine Triphosphatases; D002118:Calcium