Enzymatic Properties of Human Na,K-ATPase α1β3Isozyme

Recent results of a wide-scale human cDNA sequencing project have identified a cDNA which encodes a hitherto unknown human protein sequence exhibiting structural similarities with β-subunits of the Na,K- and H,K-ATPase family and with the amphibian Na,KATPase β3-subunit, in particular. In this study the ability of the putative human β3-subunit to assemble with the human α1-subunit in functionally active Na,KATPase was examined using the baculovirus expression system. The recombinant baculovirus simultaneously expressing both α1and β3human proteins was produced using the dual-promoter transfer vector p2Bac. The expression of both human proteins in baculovirus-infected Sf-9 cell membranes detected with specific antibodies resulted in the formation of a catalytically competent α1β3ATPase complex. Characterization of the recombinant ATPase complex involved the analysis of Na+, K+, and ATP dependencies of enzyme activity and its sensitivity toward ouabain. Preparations of HeLa cell membranes containing α1β1isozyme of human Na,K-ATPase were used as control. The data obtained clearly demonstrated that α1β3ATPase exhibits enzymatic properties which are characteristic of Na,K-ATPase. The recombinant α1β3isozyme displayed significantly lower sensitivity to ouabain than native α1β1. These findings indicate that the hitherto unknown α1β3isozyme of human Na,K-ATPase is likely to existin vivo,thus suggesting further expansion of human Na,K-ATPase isozyme diversity. The present studies are the first in which heterologous expression has been used for the characterization of an isozyme of human Na,K-ATPase.