Transfection of Na,K-ATPase alpha-subunit: regulation of enzyme abundance.
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 alpha 1-submit 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 alpha 1 for the endogenous subunit in the functional enzyme. Immunoblotting with specific antibodies confirmed the similarities in overall alpha abundance between control and transfected cells. Hybridization analysis of total RNA, however, revealed a higher abundance of the mRNA encoding total alpha 1 in transfected cells. The results suggest that endogenous and introduced alpha-submit compete for a limited amount of beta, with rapid degradation of unassembled subunits.
Shanbaky
N M
NM
Department of Integrative Biology, University of Texas Medical School, Houston 77030, USA.
Pressley
T A
TA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D000882:Haplorhini; D008969:Molecular Sequence Data; D010042:Ouabain / Q000378:metabolism; D012313:RNA / Q000032:analysis*; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*
D010042:Ouabain; D012313:RNA; D000254:Sodium-Potassium-Exchanging ATPase