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Erythrocyte Ca2+-ATPase: Activation by Enzyme Oligomerization Versus by Calmodulin

  • D. Kosk-Kosicka
  • T. Bzdega
  • A. Wawrzynow
  • S. Scaillet
  • K. Nemcek
  • J. D. Johnson
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 269)

Summary

The subject of our studies is the mechanism of activation of the erythrocyte Ca2+-ATPase. Using purified, detergent solubilized enzyme it was found that equivalent maximal Ca2+-ATPase activity is obtained either upon addition of calmodulin or upon increase of enzyme concentration. Three independent methods, including Ca20+ -ATPase activity, polarization of the enzyme modified with an external fluorescent probe, and efficiency of fluorescence resonance energy transfer between enzyme molecules have established that the concentration dependent activation is due to enzyme oligomerization. The oligomers bind calmodulin with a lower stoichiometry (0.5 mol calmodulin/mol Ca2+-ATPase), higher Ca2+ affinity (KCa = pCa 7.4), and higher cooperativity for Ca2+ (nH = 2.6) than the monomeric form (stoichiometry =1 mol calmodulin/mol Ca2+-ATPase, KCa = pCa 7.0, nH = 1.1). The Ca2+ dependence of calmodulin binding and activation of monomers indicates that calmodulin binds before the Ca2+-ATPase activity is exhibited, demonstrating that the activation of this enzyme form is totally dependent on calmodulin. In contrast, oligomers reveal very similar Ca2+ dependence for calmodulin binding and for Ca2+-ATPase activity as well as for Ca2+ binding (assessed by tryptophan fluorescence), and for the oligomerization process (assessed by fluorescence energy transfer). The calmodulin antagonist drug 48/80 inhibits the calmodulin dependent activity of the monomers (I50=1.4 μg/ml) but has no effect on the activity of oligomers, confirming that calmodulin plays no role in the activation of the oligomeric enzyme. Our studies indicate that the erythrocyte Ca2+-ATPase can be activated by its high affinity, Ca2+ dependent binding of calmodulin or by a Ca2+ dependent oligomerization process which may involve calmodulin binding site.

The red cell Ca2+-ATPase has been studied as the most experimentally accessible of the calmodulin regulated plasma membrane Ca2+-ATPascs (Schatzmann, 1982; Carafoli and Zurini, 1982). In the red cell the enzyme serves as the only Ca extrusion system to maintain intracellular Ca2+ levels near 0.1 µM. Maintenance of this low Ca2+ level is important for normal physiological functions. Further, perturbation of intracellular Ca2+ has been linked to the etiology of certain disease states, such sickle cell or hypertension (AlJaboree et al., 1984; Penniston, 1984; Vincenzi et al., 1987).

The importance of regulation of intracellular Ca2+ is reflected in the complexity of regulation of the Ca2+-ATPase. In addition to the well established stimulation of the Ca2+-ATPase activity by calmodulin (see Schatzmann, 1982) we have recently discovered that the Ca2+-ATPase is regulated by enzyme oligomerization (Kosk-Kosicka and Bzdega, 1988). Further, there is evidence for regulation by cAMP dependent phosphorylation (Neyses et al., 1985), proteolysis (Taverna et al., 1980; Enycdi et al., 1987), and acidic phospholipids (Niggli et al., 1981; Choquette et al., 1984). It is the goal of this work to elucidate the mechanism of Ca2+-ATPase regulation by calmodulin and by oligomerization, in order to eventually understand the in vivo regulation of the enzyme in health and disease.

Keywords

ATPase Activity Enzyme Concentration Fluorescence Resonance Energy Transfer Malachite Green Enzyme Molecule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • D. Kosk-Kosicka
    • 1
  • T. Bzdega
    • 1
  • A. Wawrzynow
    • 1
  • S. Scaillet
    • 1
  • K. Nemcek
    • 2
  • J. D. Johnson
    • 2
  1. 1.Department of Biological ChemistryUniversity of Maryland, School of MedicineBaltimoreUSA
  2. 2.Department of Physiological ChemistryThe Ohio State University, Medical CenterColumbusUSA

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