Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase.

The Journal of Biological Chemistry
H G SwartsJ J de Pont

Abstract

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the alpha- and beta-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1, 2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild ...Continue Reading

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Citations

May 23, 2001·Archives of Biochemistry and Biophysics·S J RulliE C Rabon
Jun 26, 1998·The EMBO Journal·H G SwartsJ J De Pont
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