Functional expression of rat alpha 1 Na,K-ATPase containing substitutions for cysteines 454, 458, 459, 513 and 551.
Site-specific mutagenesis was used to prepare the following substitutions of the rat alpha 1 Na,K-ATPase: Cys 513-->Ala, Cys 454+458+459-->Ala and Cys 551-->Ser. Ouabain-resistant HeLa cell cells expressing rat alpha 1 wild type and each of the mutants were selected, indicating that the Cys-substituted enzymes are active. Membranes isolated from HeLa cells expressing wild-type and mutant cDNAs all exhibit ouabain-insensitive Na,K-ATPase activity. The apparent K0.5 for ATP of the wild-type, Cys454+458+459-->Ala, and Cys551-->Ser enzymes are similar, while that of the Cys513-->Ala enzyme is 2.6-fold higher. These results suggest that either Cys454--Cys458 and Cys513--Cys551 are not involved in disulfide bonds, as recently suggested by Gevondyan et al (Biochem. Mol. Biol. Int. 29, 327-337, 1993), or that these two disulfides in the ATP binding region of the alpha subunit are not required for Na,K-ATPase function.
Lane
L K
LK
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D003545:Cysteine / Q000737:chemistry*; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D012694:Serine / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D012694:Serine; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D000409:Alanine