Comparison of the red blood cell Ca2(+)-ATPase in ghost membranes and after purification.
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 membraneous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca2(+)-ATPase.
Kosk-Kosicka
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University of Maryland, School of Medicine, Department of Biological Chemistry, Baltimore 21201.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D003902:Detergents; D004910:Erythrocyte Membrane / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D013861:Thiocyanates
D002147:Calmodulin; D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D011188:Potassium; D002118:Calcium