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Title: | Identification and partial purification of (Ca2+ or Mg2+)-ATPase in renal brushborder membranes | Authors: | VANERUM, M LEMMENS, Raf Berden, J. VANDUFFEL, Luc TEUCHY, Henri |
Issue Date: | 1995 | Publisher: | SPRINGER VERLAG | Source: | EUROPEAN JOURNAL OF BIOCHEMISTRY, 227(1-2). p. 150-160 | Abstract: | The protein responsible for the (Ca2+ or Mg2+)-ATPase activity in brush-border membranes from pig kidney tubular cells was characterized to distinguish this enzyme from the N-ethylmaleimide-sensitive Mg2+-ATPase, also present in renal brush borders. Both enzymes are clearly different in their pH optimum and their sensitivity to divalent cations, nucleoside 5'-triphosphates and inhibitors. Solubilization of the (Ca2+ or Mg2+)-ATPase from brush-border membrane vesicles was accomplished with Nonidet P-40 or dodecylmaltoside. However, simultaneous inactivation of the enzyme was inevitable. A tenfold enrichment of the ATPase activity was obtained by chromatofocusing of Nonidet-P-40-solubilized brush borders. A similar degree of purification was achieved by ion-exchange chromatography of dodecylmaltoside-solubilized preparations. From the SDS/polyacrylamide gels of partially purified (Ca2+ or Mg2+)-ATPase, a few protein bands could still be tentatively identified as responsible for the enzyme activity. Labeling of solubilized brush-border preparations with several radioactive ATP analogues also revealed that a protein band of molecular mass 90 kDa is the most probable candidate for the catalytic peptide of the (Ca2+ or Mg2+)-ATPase. Finally, immunoprecipitation as well as semi-dry blotting with antibodies generated against partially purified enzyme preparations, confirmed that a 90-kDa component is a reasonable candidate for the (Ca2+ or Mg2+)-ATPase in renal brush-border membranes. | Notes: | LIMBURGS UNIV CENTRUM,DEPT MBW,BIOCHEM LAB,B-3590 DIEPENBEEK,BELGIUM. UNIV AMSTERDAM,EC SLATER INST BIOCHEM RES,AMSTERDAM,NETHERLANDS. | Keywords: | ATPASE, (CA2+ OR MG2+); ECTO-ATPASE; KIDNEY, BRUSH BORDER | Document URI: | http://hdl.handle.net/1942/3640 | Link to publication/dataset: | ttp://dx.doi.org/10.1111/j.1432-1033.1995.tb20371.x | ISI #: | A1995QB85100018 | Type: | Journal Contribution |
Appears in Collections: | Research publications |
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