Abstract
We have compared properties of the red blood cell Ca2+-ATPase in two types of preparations: red cell membrane ghosts (enzyme in unfractionated membranes) and after purification (detergent-soluble enzyme). The Ca2+-ATPase activity was studied with respect to its requirement for: calmodulin, calcium, magnesium, monovalent cations, ionic strength, pH, and temperature. Sensitivity of the Ca2+-ATPase activity in the two preparations to anticalmodulin drugs and to engineered calmodulins with amino acid substitutions was determined. Finally, stoichiometry of the formation of phosphorylated enzyme intermediate (EP) and titrations of the ATP binding region with fluorescein 5′-isothiocyanate (FITC) were characterized. For the first time a high phosphorylation level of 2.0–2.4 mmol EP/mg of purified enzyme is reported.
The two enzyme preparations have been found to be very similar with respect to the dependency of all the regulating factors described here. These results complement findings reported from various laboratories on the similarity of other kinetic properties as well as the similarity of modulation of the Ca2+-ATPase activity by phospholipids and proteolysis in the membranous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca2+-ATPase.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Niggli V, Penniston J, Carafoli E: Purification of the (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J Biol Chem 254: 9955–9958, 1979
Gietzen K, Tejcka M, Wolf HU: Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca2+ + Mg2+)-dependent adenosine triphosphatase. Biochem J 189: 81–88, 1980
Penniston JT: Plasma membrane Ca2+-ATPases as active Ca2+ pumps. In: Calcium and cell function vol. 4. Academic Press, New York, 1983, pp 99–149
Filoteo AG, Gorski JP, Penniston JT: The ATP-binding site of the erythrocyte membrane Ca2+-pump. J Biol Chem 262: 6526–6530, 1987
James P, Maeda M, Fisher R, Verma AK, Krebs J, Penniston JT, Carafoli E: Identification and primary structure of a calmodulin binding domain of the Ca2+-pump of human erythrocytes. J Biol Chem 263: 2905–2910, 1988
Kosk-Kosicka D, Inesi G: Cooperative calcium binding and calmodulin regulation in the calcium-dependent adenosine triphosphatase purified from the erythrocyte membrane. FEBS Lett 189: 67–71, 1985
Kosk-Kosicka D, Scaillet S, Inesi G: The partial reactions in the catalytic cycle of the calcium dependent adenosinetriphosphatase purified from the erythrocyte membrane. J Biochem 261: 3333–3338, 1986
Kosk-Kosicka D, Bzdega T: Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin. J Biol Chem 263: 18184–18189, 1988
Kosk-Kosicka D, Bzdega T, Wawrzynow A: Fluorescence energy transfer studies of purified erythrocyte Ca2+-ATPase: Ca2+ regulated activation by oligomerization. J Biol Chem 264: 19495–19499, 1989
Chiesi M, Zurini M, Carafoli E: ATP synthesis catalyzed by the purified erythrocyte Ca-ATPase in the absence of calcium gradients. Biochemistry 25: 2595–2600, 1984
Krebs J, Vasak M, Scarpa A, Carafoli E: Conformational differences between the E1 and E2 states of the calcium adenosinotriphosphatase of the erythrocyte plasma membrane as revealed by circular dichroism and fluorescence spectroscopy. Biochemistry 26: 3921–3926, 1987
Carafoli E, Zurini M: The Ca 2+ pumping ATPase of plasma membranes. Purification, reconstitution and properties. Biochim Biophys Acta 683: 279–301, 1982
Rega AF, Garrahan PJ: The Ca2+ pump of plasma membranes. CRC Press Inc, Boca Raton, FL. 1986
Niggli V, Adunyah ES, Penniston JT, Carafoli E: Purified (Ca 2+-Mg2+)-ATPase of the erythrocyte membrane. J Biol Chem 256: 395–401, 1981
Kosk-Kosicka D, Bzdega T, Wawrzynow A, Scaillet S, Nemcek K, Johnson JD: Erythrocyte Ca2+-ATPase: activation by enzyme oligomerization versus by calmodulin. In: DEM Lawson and R Pochet (eds) Calcium binding proteins in normal and transformed cells. Plenum Press, New York, 1990
Graf E, Verma AK, Gorski JP, Lopaschuk G, Niggli V, Zurini M, Carafoli E, Penniston JT: Biochemistry 21: 4511–4516, 1982
Scharff O: Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes. Biochim Biophys Acta 512: 309–317, 1978
Mollman JE, Pleasure DE: Calcium transport in human inside-out erythrocyte vesicles. J Biol Chem 255: 569–574, 1980
Wins P, Schoffeniels E: Studies on red-cell ghost ATPase systems: properties of a (MG2+ + Ca2+)-dependent ATPase. Biochim Biophys Acta 120: 341, 1966
Scharff O: Regulation of (Ca2+, Mg2+)-ATPase in human erythrocytes dependent on calcium and calmodulin. Acta Biol Med Germ 40: 457–461, 1981
Kosk-Kosicka D, Bzdega T: Effects of calmodulin on erythrocyte Ca2+-ATPase activation and oligomerization. Biochemistry 29: 3772–3777, 1990
Gietzen K, Adamczyk-Engelman P, Wuthrich A, Konstantinova A, Bader H: Compound 48/80 is a selective and powerful inhibitor of calmodulin-regulated functions. Biochim Biophys Acta 736: 109–118, 1983
Craig TA, Watterson DM, Prendergast FG, Haiech J, Roberts DM: Site-specific mutagenesis of the α-helices of calmodulin. J Biol Chem 262: 3278–3284, 1987
Muallem S, Karlish SJD: Catalytic and regulatory ATPbinding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate. J Biol Chem 258: 169–175, 1983
Andersen JP, Moller JV, Jorgensen PL: The functional unit of sarcoplasmic reticulum Ca2+-ATPase, Active site titration and fluorescence measurements. J Biol Chem 257: 8300–8307, 1982
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kosk-Kosicka, D. Comparison of the red blood cell Ca2+-ATPase in ghost membranes and after purification. Mol Cell Biochem 99, 75–81 (1990). https://doi.org/10.1007/BF00230336
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00230336