Transfection of Na,K-ATPase α-subunit: regulation of enzyme abundance

Abstract

DNA-mediated gene transfer into mammalian cells was used as a model for investigating the regulation of Na,K-ATPase abundance. Complementary DNA encoding the catalytic α1-subunit from rat was introduced into ouabain-sensitive monkey kidney cells, and transfectants were selected by their ability to survive in normally cytotoxic concentrations of ouabain. The overall specific activity of Na,K-ATPase in the membranes of transfectants was not significantly different from that in control cells, suggesting that there was a partial replacement, rather than an addition, of introduced α1for the endogenous subunit in the functional enzyme. Immunoblotting with specific antibodies confirmed the similarities in overall α abundance between control and transfected cells. Hybridization analysis of total RNA, however, revealed a higher abundance of the mRNA encoding total α1in transfected cells. The results suggest that endogenous and introduced α-subunit compete for a limited amount of β, with rapid degradation of unassembled subunits.Key words: DNA-mediated gene transfer, immunoblots, protein abundance, subunit assembly.