Specific modification of gastric K+-stimulated ATPase activity by thimerosal |
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Authors: | John G Forte Jean L Poulter Ross Dykstra Jose Rivas |
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Affiliation: | Department of Physiology-Anatomy, University of California, Berkeley, CA 94720 U.S.A. |
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Abstract: | Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K+-ATPase and the K+-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K+-stimulated ATPase, while K+-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K+-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K+-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K+-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; Km for p-nitrophenylphosphate unchanged for Ka for K+ increased. ATP, which is a potent inhibitor of K+-p-nitrophenylphosphatase activity in native membranes (KI ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K+-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K+-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H+-transport reaction. |
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Keywords: | Sulfhydryl reagent Thimerosal Membrane modification Secretion Pig stomach Pipes piperazine-N N′-bis(ethanesulfonic acid) |
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