Conformational changes of the in situ red cell membrane calcium pump affect its proteolysis.
In inside-out red cell membrane vesicles trypsin digestion reduces the molecular mass of the 32P-labeled acyl-phosphate intermediate of the calcium pump from the original 140 kDa to about 80 kDa with a simultaneous activation of the calcium uptake. This process is slightly stimulated by the presence of calcium, as compared to EGTA, or EGTA + vanadate, but the proteolytic pattern is similar under all these conditions. However, trypsin degradation of the 80 kDa polypeptide, resulting in the loss of calcium transport activity and 32P-phosphoenzyme formation, is rapid in the presence of calcium, inhibited by EGTA and almost fully blocked by EGTA + vanadate. In the presence of these latter ligands, probably locking the calcium pump in an E2 conformation, the 80 kDa protein becomes insensitive even to excessive digestion by the non-specific protease, pronase. The data indicate major changes in the molecular arrangement of the calcium pump protein when transformed from a calcium-liganded (E1) to an E2 conformation.
D000252:Calcium-Transporting ATPases / Q000097:blood*; D004533:Egtazic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D011402:Pronase; D011487:Protein Conformation; D014357:Trypsin; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D014639:Vanadium; D014638:Vanadates; D004533:Egtazic Acid; D014357:Trypsin; D011402:Pronase; D000252:Calcium-Transporting ATPases