Glutamate 329 located in the fourth transmembrane segment of the .alpha.-subunit of the rat kidney sodium-potassium-ATPase is not an essential residue for active transport of sodium and potassium ions
In: Biochemistry, Jg. 32 (1993-12-01), S. 13340-13349
Online
unknown
Zugriff:
An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G to C base substitution in the cDNA, which on the amino acid level gave rise to a glutamine in place of the glutamate residue Glu329, previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. Functional analysis on isolated plasma membranes demonstrated that the Glu329-->Gln variant was able to catalyze Na(+)- and K(+)-activated ATPase activity with a maximum turnover number similar if not identical to that of the wild type, but the variant exhibited a reduced affinity for both cations corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold increase in K0.5 for K+. Moreover, the apparent affinity for ATP was increased 15-fold in the variant relative to wild-type Na+,K(+)-ATPase. The Na+,K(+)-ATPase activity of the variant displayed an anomalous pH dependence with a down-shift of the pH optimum and a nearly constant rate in the range between pH 7.0 and 8.7.
Titel: |
Glutamate 329 located in the fourth transmembrane segment of the .alpha.-subunit of the rat kidney sodium-potassium-ATPase is not an essential residue for active transport of sodium and potassium ions
|
---|---|
Autor/in / Beteiligte Person: | Vilsen, Bente |
Link: | |
Zeitschrift: | Biochemistry, Jg. 32 (1993-12-01), S. 13340-13349 |
Veröffentlichung: | American Chemical Society (ACS), 1993 |
Medientyp: | unknown |
ISSN: | 1520-4995 (print) ; 0006-2960 (print) |
DOI: | 10.1021/bi00211a048 |
Schlagwort: |
|
Sonstiges: |
|