Gastric HCO3-stimulated ATPase: Evidence against its microsomal localization in rat fundus mucosa |
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Authors: | Annick Soumarmon Miguel Lewin Anne Marie Cheret Serge Bonfils |
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Affiliation: | Unité de Recherches de Gastroentérologie, INSERM U. 10, Hôpital Bichat, 170 Bd Ney, F. 75877 Paris Cedex 18 France |
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Abstract: | Rat gastric mucosa was shown to contain a Mg2+-dependent ATPase which is stimulated by HCO3− at pH 8–9.Triton X-100 solubilizes this HCO3−-stimulated, Mg2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3).The gastric mucosa was resolved into five subcellular fractions by differential centrifugation. A large granule fraction (Fraction M), 28 000 g · min, was characterized by cytochrome c oxidase (marker enzyme for mitochondria). A microsomal fraction (Fraction P), 2 760 000 g · min, was characterized by 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) (plasma membrane).The Mg2+-dependent ATPase was demonstrated to have a bimodal mitochondrial membranous localization: 24% of its activity is associated with cytochrome c oxidase, and 75% with 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) at pH 8.The HCO3− addition resulted in two opposite effects: (1) a strong stimulation (84%) in Fraction M; (2) a slight inhibition (12%) in Fraction P.Fraction M was subfractionated by equilibration on a sucrose gradient. It gave rise to a homogeneous mitochondrial (d, 1.17–1.21) Mg2+-dependent ATPase, closely associated with cytochrome c oxidase. This ATPase is strongly stimulated (×2) by HCO3−. The subfractionation of Fraction P gave rise to two distinct ATPases: (1) the major one is associated with membranous (d, 1.10–1.15) material marked by 5′-nucleotidase and is slightly inhibited by HCO3−; (2) the other is associated with denser (d, 1.17–1.21) material and is stimulated by HCO3−.The bicarbonate-stimulated fraction of the Mg2+-dependent ATPase activity found in the gastric microsomal fraction is assumed to arise from mitochondrial cross-contamination. Further support comes from the optimal HCO3− concentration. In addition, SCN− is shown to specifically inhibit the ATPase of Fraction M.From these results it appears that the implication of HCO3−-stimulated ATPase in the gastric secretion of H+ is not as clear as had been suggested. However, in the view of an ATPase-supported model for H+ secretion, attention can be directed towards the Mg2+-dependent ATPase found to be associated with microsomes. |
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