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J. Biol. Chem., Vol. 263, Issue 16, 7726-7733, 06, 1988
JR Emanuel, J Schulz, XM Zhou, RB Kent, D Housman, L Cantley and R Levenson
We have used a gene transfer system to investigate the relationship between
expression of the rat Na,K-ATPase alpha 1 subunit gene and
ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire
rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector
pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM
ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of
transfected clones revealed the presence of both rat- specific and
endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single
form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells,
whereas two distinct classes of ouabain- inhibitable uptake were observed
in transfectants. One class exhibited the high ouabain sensitivity of the
endogenous monkey Na,K-ATPase, while the second class showed the reduced
ouabain sensitivity characteristic of the rodent renal Na,K-ATPase.
Examination of the ouabain-sensitive, sodium-dependent ATPase activity of
the transfectants also revealed a low affinity component of Na,K-ATPase
activity characteristic of the rodent kidney enzyme. These results suggest
that expression of the rat alpha 1 subunit gene is directly responsible for
ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.
Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
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