The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid.
Peptide G25 (KKAVKVPKKEKSVLQGKLTRLAVQI) representing the putative lipid-binding region (G region) of the erythrocyte Ca2+ pump was synthesized. This peptide interacted with acidic lipids, as shown by the increase in size of phosphatidylserine liposomes in its presence. This lipid interaction is consistent with the previous evidence suggesting that the portion of the pump from which this peptide was taken is responsible for the activation of the pump by acidic lipid. G25 also bound to calmodulin, as was shown by its cause of a shift in the fluorescence of 5-dimethylamino naphthalene-1-sulfonyl- (dansyl)-calmodulin, and by its competition with Ca2+ pump for calmodulin. Its Kd for dansyl-calmodulin was much higher (0.8 microM) than that of the peptides representing the primary calmodulin-binding region (C region) of the Ca2+ pump. Although the presence of the G region provided the possibility of a second calmodulin-binding site, activation of the pump by calmodulin always could be fitted by simple saturation kinetics. The calmodulin-binding peptide from the C region of the pump, C28R2, also interacted with lipid with even greater effectiveness than G25. When the C region of the pump was saturated with calmodulin, acidic lipid activation of the pump followed simple saturation kinetics. However, when calmodulin was omitted, a higher concentration of lipid was needed for saturation and the kinetics became complex. The data are consistent with the idea that calmodulin activates the pump only by interaction at the C region, but that acidic lipid activates by interaction at both of the C and G regions.
Filoteo
A G
AG
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
Enyedi
A
A
Penniston
J T
JT
D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000031:analogs & derivatives / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004910:Erythrocyte Membrane / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D050356:Lipid Metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
C035684:5-dimethylaminonaphthalene-1-sulfonyl-calmodulin; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008081:Liposomes; D008565:Membrane Proteins; D010718:Phosphatidylserines; C075126:calmodulin-binding peptide G25; D000252:Calcium-Transporting ATPases