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Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes
- Source :
- The Biochemical journal. 269(3)
- Publication Year :
- 1990
-
Abstract
- 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.
- Subjects :
- Intracellular Fluid
GTP'
Sodium
chemistry.chemical_element
Guanosine
Digitonin
Wasp Venoms
Biology
Biochemistry
Ouabain
Exocytosis
Sodium Channels
chemistry.chemical_compound
Xenopus laevis
Adenosine Triphosphate
Calmodulin
medicine
Animals
Protease Inhibitors
Molecular Biology
Ovum
Polymyxin B
Manganese
Binding Sites
Ionophores
Nucleotides
Cell Membrane
Neomycin
Cell Biology
Adenosine
Stimulation, Chemical
beta-N-Acetylhexosaminidases
Culture Media
chemistry
Mastoparan
Ionomycin
Oocytes
Potassium
Intercellular Signaling Peptides and Proteins
Calcium
Peptides
medicine.drug
Research Article
Subjects
Details
- ISSN :
- 02646021
- Volume :
- 269
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- The Biochemical journal
- Accession number :
- edsair.doi.dedup.....dea646a7546260cc1648e2d6f5044fca