Different conformational states of the purified Ca2+-ATPase of the erythrocyte plasma membrane revealed by controlled trypsin proteolysis.
The purified Ca2+-pumping ATPase of the erythrocyte membrane has been exposed to trypsin at 37 degrees C, in the presence of different effectors of its activity. The control proteolytic pattern is characterized by a number of transient and of limit polypeptides (Zurini, M., Krebs, J., Penniston, J. T., and Carafoli, E. (1984) J. Biol. Chem. 259, 618-627). The effectors influence the pattern in the Mr region 90,000-76,000, which contains the calmodulin binding domain and the active site of the enzyme. In this region, polypeptides of 90, 85, 81, and 76 kDa are clearly visible in the controls. 1) Calmodulin plus Ca2+ induces the faster disappearance of the 90-kDa product and the relative accumulation of the 85-kDa with respect to the 81-kDa polypeptide. 2) Vanadate plus Mg2+ also accelerates the disappearance of the 90-kDa product. However, they induce the relative accumulation of the 81-kDa polypeptide. 3) Linoleic acid, which stimulates the activity of the enzyme to the same levels obtained with calmodulin, greatly accelerates the rate of trypsin proteolysis, causing the virtual disappearance of all polypeptides in the 90-76-kDa region. 4) The 81-kDa polypeptide has maximal ATPase activity and is insensitive to calmodulin; the 85-kDa polypeptide has lower ATPase activity and binds calmodulin, but is not stimulated (or is stimulated only negligibly) by the activator.
D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D002147:Calmodulin / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D002147:Calmodulin; D008041:Linoleic Acids; D010446:Peptide Fragments; D014639:Vanadium; D014638:Vanadates; D019787:Linoleic Acid; D014357:Trypsin; D000252:Calcium-Transporting ATPases