Structure--function relationship of the human erythrocyte plasma membrane Ca(2+)-ATPase revealed by V8 protease treatment.
Treatment of the solubilized and purified Ca(2+)-translocating ATPase (Ca(2+)-ATPase) (136 kDa) from human erythrocyte plasma membranes with endoproteinase Glu-C from Staphylococcus aureus strain V8 (V8 protease) yielded transient fragments of 96 kDa and 76 kDa and more stable fragments of 60 kDa and 37/36 kDa (doublet). The presence of calmodulin did not alter the fragmentation pattern. The 60 kDa fragment contains the protein kinase C (bovine brain) phosphorylation site(s), which we previously localized in the C-terminal region [Wang, Wright, Machan, Allen, Conigrave & Roufogalis (1991) J. Biol. Chem. 266, 9078-9085]. On the other hand, the 37/36 kDa fragments possess the ability to form an acyl-phosphate intermediate. Furthermore, the presence of the 60 kDa and 37/36 kDa fragments together results in expression of calmodulin-sensitive Ca(2+)-ATPase activity. However, further degradation of the 60 kDa fragment was coupled with the appearance of calmodulin-independent activity, whereas the 37/36 kDa fragment doublet remained stable. It was concluded that the 60 kDa and the 37/36 kDa fragments: (a) together represent the C-terminal two-thirds of the enzyme, which is functional as an Ca(2+)-ATPase, (b) were produced by a single cleavage near the C-terminal side of the cytosolic catalytic domain, and (c) probably remain physically and functionally associated even after cleavage has occurred. At the C-terminus, the basic calmodulin-binding domain is flanked by two highly acidic regions (domains A and B). Our results indicate that domains A and B, despite containing many Asp and Glu residues, were not readily cleaved by V8 protease, which is known to cleave selectively peptide bonds at the C-terminal side of Asp and Glu. However, if the Ca(2+)-ATPase were pre-digested with calpain I from human erythrocytes, which removed its calmodulin-binding domain (along with domain B), multiple cleavages by V8 protease in domain A were then readily observed. We propose that the calmodulin-binding domain is closely associated with the acidic domains A and B and that these acidic domains might help to co-ordinate the stimulation of the enzyme by calmodulin.
Wang
K K
KK
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Roufogalis
B D
BD
Kuo
T H
TH
D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship
D002147:Calmodulin; D010446:Peptide Fragments; D011493:Protein Kinase C; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002154:Calpain; D000252:Calcium-Transporting ATPases