A GLU(329)-] GLN VARIANT OF THE ALPHA-SUBUNIT OF THE RAT-KIDNEY NA-ATPASE CAN SUSTAIN ACTIVE-TRANSPORT OF NA+ AND K+ AND NA+,K+-ACTIVATED ATP HYDROLYSIS WITH NORMAL TURNOVER NUMBER(,K+)
B. Vilsen, A GLU(329)-] GLN VARIANT OF THE ALPHA-SUBUNIT OF THE RAT-KIDNEY NA-ATPASE CAN SUSTAIN ACTIVE-TRANSPORT OF NA+ AND K+ AND NA+,K+-ACTIVATED ATP HYDROLYSIS WITH NORMAL TURNOVER NUMBER(,K+), FEBS letters, 333(1-2), 1993, pp. 44-50
An allelic variant of the ouabain-insensitive rat kidney Na+,K+-ATPase
alpha1-isoform was identified by chance in a cDNA library. The varian
t differed from the wild-type rat kidney Na+,K+-ATPase by a single G-t
o-C base substitution in the cDNA, which on amino acid level gave rise
to a glutamine in place of the glutamate residue Glu329 previously su
ggested as a likely donator of oxygen ligands for Na+ and K+ binding.
The variant cDNA was transfected into COS-1 cells and the transfectant
s expanded with success into stable cell lines that were able to grow
in the presence of a concentration of ouabain highly cytotoxic to the
parental cells containing only the endogenous COS-1 cell Na+,K+-ATPase
. Under these conditions, the viability of the cells depended on the c
ation transport mediated by the ouabain-insensitive Glu329-->Gln varia
nt, whose cDNA was shown by polymerase chain reaction amplification to
be stably integrated into the COS-I cell genome. The maximum specific
ATP hydrolysis activity of isolated plasma membranes of the Glu329-->
Gln variant did not differ significantly from that of plasma membranes
containing the wild type. A method was established for measurement of
the phosphorylation capacity of the expressed Glu329-->Gln variant an
d wild-type enzyme, and it was thereby demonstrated that the variant h
ad a turnover number similar if not identical to that of the wild-type
.