Photochemical cross-linking of azidocalmodulin to the (Ca2+ + Mg2+)-ATPase of the erythrocyte membrane.
The calmodulin (CaM)-binding proteins present in the human red blood cell membrane were examined through photoaffinity labeling. Two different membrane preparations were used: white ghosts and inside-out vesicles. These were incubated with azido-125I-CaM, a photoactivatable derivative of calmodulin, and photolyzed. Cross-linked products were identified by autoradiography of dried sodium dodecyl sulfate slab gels and quantitated by counting gel slices for 125I. The major product formed with each membrane type had an apparent Mr of 168,000. No other product was common to both membrane types; however, a few other products unique to each vesicle type were detected. The formation of the 168,000-dalton product correlated with an increased basal (Ca2+ + Mg2+)-ATPase activity in white ghosts and with an increased basal Ca2+ transport in inside-out vesicles. This suggests that the 168,000-dalton product represents a cross-link between azido-125I-CaM and the Ca2+ pump ATPase. The ATPase appears to be a single protein of about 150,000 daltons, which can bind a single calmodulin. No evidence was found to indicate anything other than a 1:1 binding stoichiometry between calmodulin and the (Ca2+ + Mg2+)-ATPase.
D001386:Azides; D017301:Ca(2+) Mg(2+)-ATPase; D002135:Calcium-Binding Proteins / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000031:analogs & derivatives / Q000097:blood*; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010777:Photochemistry; D011485:Protein Binding
D001386:Azides; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D003432:Cross-Linking Reagents; C030955:azidocalmodulin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases