Abstract
The purified plasma membrane Ca2+-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2+ concentrations (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189; Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). We have previously shown that the Ca2+-ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding. Presently, we have succeeded in determining the molecular mass of the two active enzyme species by equilibrium ultracentrifugation. For the calmodulin-dependent species, the molecular mass is 170 +/- 30 kDa, which is consistent with predominantly monomeric Ca2+-ATPase with bound calmodulin. The molecular mass of calmodulin-independent oligomers is 260 +/- 34 kDa, indicating that they are dimers. Results of experiments performed under different calcium and potassium concentrations and in the presence of dextran that causes molecular crowding verify a strict Ca2+ requirement of the dimerization process. We conclude that the active species of the Ca2+-ATPase are a monomer-calmodulin complex and a dimer.
Highlights
The purified plasma membrane Ca2؉-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2؉ concentrations (Kosk-Kosicka, D., and Bzdega, T. (1988) J
We have previously shown that the Ca2؉-ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding
We undertook the determination of the molecular mass of different forms of the erythrocyte Ca2ϩ-ATPase, the oligomeric form with calmodulin-independent activity and the form with calmodulin-dependent activity previously termed monomer
Summary
The purified plasma membrane Ca2؉-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2؉ concentrations (Kosk-Kosicka, D., and Bzdega, T. (1988) J. The purified plasma membrane Ca2؉-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2؉ concentrations We have previously shown that the Ca2؉-ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding. For the calmodulin-dependent species, the molecular mass is 170 ؎ 30 kDa, which is consistent with predominantly monomeric Ca2؉-ATPase with bound calmodulin. We conclude that the active species of the Ca2؉-ATPase are a monomer-calmodulin complex and a dimer. Using measurements of fluorescence resonance energy transfer between appropriately labeled enzyme molecules, we were able to differentiate two active species of the enzyme, which we defined as oligomers and monomers with bound calmodulin [2]. The extent of oligomerization and the structure of the active forms of the enzyme could not be determined by the methods used previously, and it is this point that is addressed here
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