Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin.
The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.
Kosk-Kosicka
D
D
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Bzdega
T
T
D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000494:pharmacology*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D013861:Thiocyanates
D002147:Calmodulin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate