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Mimicking of K+ activation by double mutation of glutamate 795 and glutamate 820 of gastric H+,K+-ATPase.
Mimicking of K+ activation by double mutation of glutamate 795 and glutamate 820 of gastric H+,K+-ATPase.
- Source :
- Biochemistry; 6527; 6533; 0006-2960; 21; 40; ~Biochemistry~6527~6533~~~0006-2960~21~40~~
- Publication Year :
- 2001
-
Abstract
- Item does not contain fulltext<br />Six double mutants of Glu(795) and Glu(820) present in transmembrane domains 5 and 6 of the alpha-subunit of rat gastric H(+),K(+)-ATPase were generated and expressed with the baculovirus expression system. Five of the six mutants exhibited an SCH 28080-sensitive ATPase activity in the absence of K(+). The activity levels decreased in the following order: E795Q/E820A > E795Q/E820Q > E795Q/E820D congruent with E795A/E820A > E795L/E820Q. The E795L/E820D mutant possessed no constitutive activity. The relative low ATPase activity of the E795L/E820Q mutant is due to its low phosphorylation rate so that the dephosphorylation step was no longer rate-limiting. The constitutively active mutants showed a much lower vanadate sensitivity than the wild-type enzyme and K(+)-sensitive mutants, indicating that these mutants have a preference for the E(1) conformation. In contrast to the constitutively active single mutants generated previously, the double mutants exhibited a high spontaneous dephosphorylation rate at 0 degrees C compared to that of the wild-type enzyme. In addition, the H(+),K(+)-ATPase inhibitor SCH 28080 increased the steady-state phosphorylation level of the constitutively active mutants, due to the formation of a stable complex with the E(2)-P form. These studies further substantiate the idea that the empty ion binding pockets of some mutants apparently mimic the K(+)-filled binding pocket of the native enzyme.
Details
- Database :
- OAIster
- Journal :
- Biochemistry; 6527; 6533; 0006-2960; 21; 40; ~Biochemistry~6527~6533~~~0006-2960~21~40~~
- Publication Type :
- Electronic Resource
- Accession number :
- edsoai.on1284016764
- Document Type :
- Electronic Resource