Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase. Ca2+-regulated activation by oligomerization.
Fluorescence resonance energy transfer has been used to study oligomerization of the purified erythrocyte Ca2+-ATPase. The energy transfer efficiency has been measured at different enzyme concentrations, from fluorescein 5'-isothiocyanate attached on one enzyme molecule to eosin 5-maleimide or tetramethylrhodamine 5-isothiocyanate attached on another enzyme molecule. The energy transfer efficiency showed a sigmoid dependence on enzyme concentration and was half-maximal at 10-12 nM enzyme; this dependence on enzyme concentration closely resembled previously demonstrated dependence of Ca2+-ATPase activity and polarization of the fluorescein 5'-isothiocyanate enzyme (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189). Thus, the three independent methods establish that enzyme concentration-dependent oligomerization is a mechanism of activation of the erythrocyte Ca2+-ATPase. Further energy transfer studies demonstrated that enzyme oligomerization required calcium. This calcium dependence was characterized by high affinity (half-maximal energy transfer at pCa 7.15) and cooperativity (Hill coefficient of 2.36), being very similar in both respects to the Ca2+ dependence of the Ca2+-ATPase activity. The data indicated that the oligomerization process produced a highly cooperative, Ca2+-regulated activation of the enzyme at physiologically relevant Ca2+ concentrations. These studies show that the Ca2+-ATPase can be fully activated by a Ca2+-dependent oligomerization mechanism, which is independent of the previously described activation by calmodulin. We propose two pathways for the activation of the Ca2+-ATPase, taking into account the interdependencies between the Ca2+, calmodulin, and enzyme concentrations.
Kosk-Kosicka
D
D
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Bzdega
T
T
Wawrzynow
A
A
D002122:Calcium Chloride / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D004735:Energy Transfer; D004910:Erythrocyte Membrane / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013861:Thiocyanates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002122:Calcium Chloride