Datensatz

Zusammenfassung

As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca²⁺ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K_0.5 0.5 microM-Ca²⁺, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca²⁺ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg²⁺ or ATP completely abolished the Ca²⁺ effect. Half-maximal stimulation by Ca²⁺ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5'-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). P_i, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5'-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5'-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca²⁺ effect by some 50%. Inhibitors of chymotrypsin and the Ca²⁺ proteinase calpain had no effect. Ca²⁺ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca²⁺ response entirely. Neomycin also abolished a Ca²⁺-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca²⁺-regulated pathway for the plasma membrane insertion of sodium pumps.