Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding alpha 3-subunits of rat brain Na+,K+-ATPase.
The cDNAs encoding alpha 3-subunits of rat brain Na+,K+-ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co-transfection method. Stably transformed cells were selected with 300 micrograms/ml of neomycin (G-418) for 6 weeks. Northern blot analysis using the 3'-non-translated region of the cDNA as a probe revealed that the alpha 3 mRNA appeared in transfected cells. Na+,K+-ATPase activity of the transfected cells was twice that of wild-type cells. Regarding ouabain sensitivity, the Na+,K+-ATPase showed two Ki values for ouabain (8 x 10(-8) and 4.5 x 10(-5) M) in transfected cells while wild-type cells displayed only the higher value. Ouabain sensitivity of Rb+ uptake also demonstrated two Ki values in the transfected cells (8 x 10(-8) and 4 x 10(-5) M) and a Ki in wild-type cells of 4 x 10(-5) M. It is concluded that alpha 3 is a highly ouabain-sensitive catalytic subunit of Na+,K+-ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the alpha-subunit rather than the beta-subunit. This is the first report on the catalytic characteristics of the alpha 3 isoform of Na+,K+-ATPase.
Hara
Y
Y
Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Japan.
Nikamoto
A
A
Kojima
T
T
Matsumoto
A
A
Nakao
M
M
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D005796:Genes; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D009693:Nucleic Acid Hybridization; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D010042:Ouabain; D004247:DNA; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium