| Popis: |
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 beta-subunits of the Na,K- and H,K-ATPase family and with the amphibian Na,KATPase beta3-subunit, in particular. In this study the ability of the putative human beta3-subunit to assemble with the human alpha1-subunit in functionally active Na,KATPase was examined using the baculovirus expression system. The recombinant baculovirus simultaneously expressing both alpha1 and beta3 human 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 alpha1beta3 ATPase 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 alpha1beta1 isozyme of human Na,K-ATPase were used as control. The data obtained clearly demonstrated that alpha1beta3 ATPase exhibits enzymatic properties which are characteristic of Na, K-ATPase. The recombinant alpha1beta3 isozyme displayed significantly lower sensitivity to ouabain than native alpha1beta1. These findings indicate that the hitherto unknown alpha1beta3 isozyme of human Na,K-ATPase is likely to exist in 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. |
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