Identification and Partial Purification of (Ca²⁺ or Mg²⁺)‐ATPase in Renal Brush‐Border Membranes

Abstract

The protein responsible for the (Ca'+ 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 (Ca'+ or Mg")-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 Mg'+)-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 90kDa is the most probable candidate for the catalytic peptide of the (Caz+ or Mg'+)-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 (Ca'+ or Mg'+)-ATPase in renal brush-border membranes.

Caracterização cinética de uma ATP-difosfohidrolase em membranas de placa óssea de rato livres de fosfatase alcalina

“…Também foi relatada a purificação parcial da enzima de fígado de rato (Lin and Fain, 1984), de células tumorais humanas (Shi and Knowles, 1994; Knowles and Leng, 1984; Gao and Knowles, 1993), de pâncreas de porco (Sévigny et al, 1995), de rim de porco (Van Erum et al, 1995), de aorta bovina (Côté et al, 1991), de pulmão de boi (Sévigny et al, 1997a), de coração de boi (Beaudoin et al, 1997) e de estômago de frango (Lewis Carl and Kirley, 1997).…”