Identification and partial purification of (Ca2+ or Mg2+)-ATPase in renal brush-border membranes.
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 Mg(2+)-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.
Limburgs Universitair Centrum, Departmente MBW, Diepenbeek, Belgium.
M
M
Van Erum
R
R
Lemmens
J
J
Berden
H
H
Teuchy
L
L
Vanduffel
D000255:Adenosine Triphosphate / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000201:enzymology*; D008871:Microvilli / Q000201:enzymology; D011233:Precipitin Tests; D013552:Swine
D000345:Affinity Labels; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases