Effects of calmodulin on erythrocyte Ca2(+)-ATPase activation and oligomerization.
The study was performed on the purified human erythrocyte Ca2(+)-ATPase to test whether or not calmodulin promotes enzyme oligomerization. Two physiologically significant modes of activation of this enzyme were considered, by calmodulin binding to monomeric enzyme and by enzyme oligomerization [Kosk-Kosicka & Bzdega (1988) J. Biol. Chem. 263, 18184]; it was not clear whether the two modes were interdependent or operated independently. Fluorescence resonance energy transfer (FRET) between separately labeled Ca2(+)-ATPase molecules was used to monitor oligomerization. No change in energy transfer efficiency was observed upon subsequent addition of calmodulin at different enzyme concentrations. Lack of decrease in the enzyme concentration at which the half-maximal oligomerization occurred indicated that calmodulin did not facilitate oligomerization. The calmodulin inhibitor compound 48/80 had no effect on either the Ca2(+)-ATPase activity of oligomers or the extent of oligomerization measured by FRET while it drastically decreased the calmodulin-stimulated activity of the monomeric Ca2(+)-ATPase. The findings demonstrate that calmodulin is not involved in the oligomerization-induced activation pathway; it neither promotes oligomerization nor stimulates the Ca2(+)-ATPase activity of oligomers. We have demonstrated that calmodulin added before mixing donor- and acceptor-labeled enzyme populations prevented the occurrence of energy transfer. This inhibition of the formation of mixed donor-acceptor enzyme oligomers by calmodulin was dose dependent. Also, the reversal of the inhibition by compound 48/80 proceeded in a dose-dependent manner. Further, calmodulin prevented the apparent decrease of energy transfer efficiency that resulted from dilution of mixed donor-acceptor enzyme oligomers with unlabeled enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Kosk-Kosicka
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D
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Bzdega
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T
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D005453:Fluorescence; D006801:Humans; D013379:Substrate Specificity
D002147:Calmodulin; D000252:Calcium-Transporting ATPases