Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes.
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 (K0.5 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). Pi, 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.
Schmalzing
G
G
Max-Planck-Institut für Biophysik, Frankfurt, Federal Republic of Germany.
Kröner
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003470:Culture Media; D004072:Digitonin / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D036341:Intercellular Signaling Peptides and Proteins; D007424:Intracellular Fluid / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010042:Ouabain / Q000378:metabolism*; D010063:Ovum / Q000187:drug effects / Q000378:metabolism; D010455:Peptides; D011112:Polymyxin B / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D015222:Sodium Channels / Q000502:physiology; D013268:Stimulation, Chemical; D014862:Wasp Venoms / Q000494:pharmacology; D014982:Xenopus laevis / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D002147:Calmodulin; D003470:Culture Media; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores; D009711:Nucleotides; D010455:Peptides; D011480:Protease Inhibitors; D015222:Sodium Channels; D014862:Wasp Venoms; D008345:Manganese; D010042:Ouabain; C037500:mastoparan; D000255:Adenosine Triphosphate; D012964:Sodium; D001619:beta-N-Acetylhexosaminidases; D009355:Neomycin; D011112:Polymyxin B; D004072:Digitonin; D011188:Potassium; D002118:Calcium