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.