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Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding α 3 ‐subunits of rat brain Na + ,K + ‐ATPase
Author(s) -
Hara Yukichi,
Nikamoto Akira,
Kojima Toshiyuki,
Matsumoto Akiko,
Nakao Makoto
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)80218-7
Subject(s) - transfection , complementary dna , microbiology and biotechnology , 3t3 cells , chemistry , sodium , biology , biochemistry , gene , organic chemistry
The cDNAs encoding α 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 μg/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 α 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 K i values for ouabain (8 × 10 −8 and 4.5 × 10 −5 M) in transfected cells while wild‐type cells displayed only the higher value. Ouabain sensitivity of Rb + uptake also demonstrated two K i values in the transfected cells (8 × 10 −8 and 4.5 × 10 −5 M) and a K i in wild‐type cells of 4 × 10 −5 M. It is concluded that α 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 α‐subunit rather than the β‐subunit. This is the first report on the catalytic charateristics of the α 3 isoform of Na + ,K + ‐ATPase.