A Glu329→ Gln variant of the α‐subunit of the rat kidney Na+K+‐ATPase can sustain active transport of Na+ and K+ and Na+K+‐activated ATP hydrolysis with normal turnover number

FEBS Letters

Vilsen, Bente¹ 

[1] Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark

关键词: Glutamate;    Allelic variant;    Na+;    K+-pump;    Cation site;    Polymerase chain reaction;    DCCD;    N;    N'-dicyclohexylcarbodiimide;    kb;    kilobase pair;    Na+K+-ATPase;    sodium plus potassium-activated adenosine triphosphatase (EC 3.6.1.3);    PCR;    polymerase chain reaction;    T m;    melting temperature;   

DOI  :  10.1016/0014-5793(93)80372-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

An allelic variant of the ouabain-insensitive rat kidney Na⁺, K⁺-ATPase α₁-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 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. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu³²⁹→ Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu³²⁹→ Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.

【 授权许可】

Unknown