In:
Biochemical Journal, Portland Press Ltd., Vol. 331, No. 2 ( 1998-04-15), p. 465-472
Abstract:
To investigate the role of Glu820, located in transmembrane domain M6 of the α-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820 → Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40–100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10–20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a] pyridine (SCH 28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH 28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH 28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the α-subunit.
Type of Medium:
Online Resource
ISSN:
0264-6021
,
1470-8728
Language:
English
Publisher:
Portland Press Ltd.
Publication Date:
1998
detail.hit.zdb_id:
1473095-9
SSG:
12
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