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  • Title: The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme.
    Author: Niggli V, Adunyah ES, Cameron BF, Bababunmi EA, Carafoli E.
    Journal: Cell Calcium; 1982 May; 3(2):131-51. PubMed ID: 6126277.
    Abstract:
    The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in a range of 30-100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4-26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adunyah, J.T. Penniston and E. Carafoli, 1981, J. Biol, Chem. 256, 395 - 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that ot the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 Ca2+/ATP hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.
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