MUTANT GLU781-]ALA OF THE RAT-KIDNEY NA-ATPASE DISPLAYS LOW CATION AFFINITY AND CATALYZES ATP HYDROLYSIS AT A HIGH-RATE IN THE ABSENCE OF POTASSIUM-IONS(,K+)
B. Vilsen, MUTANT GLU781-]ALA OF THE RAT-KIDNEY NA-ATPASE DISPLAYS LOW CATION AFFINITY AND CATALYZES ATP HYDROLYSIS AT A HIGH-RATE IN THE ABSENCE OF POTASSIUM-IONS(,K+), Biochemistry, 34(4), 1995, pp. 1455-1463
Site-specific mutagenesis was used to replace Glu329, Glu781, Asp806,
Thr809, and Asp810 in the transmembrane domain of the ouabain-insensit
ive alpha(1)-isoform of rat kidney Na+,K+-ATPase. cDNAs encoding any o
f the mutants Glu329-->Ala, Glu781-->Ala, Asp806-->Asn, Thr809-->Ala,
and Asp810-->Asn were transfected into COS cells, and transfectants we
re grown in the presence of ouabain to inhibit the endogenous COS cell
Na+,K+-ATPase. Mutants Glu781-->Ala and Thr809-->Ala were functional
as evidenced by their ability to confer ouabain resistance to the cell
s, whereas mutants Glu329-->Ala, Asp806-->Asn, and Asp810-->Asn were i
nactive. The apparent Na+ affinities determined by titrations of Na+,K
+-ATPase activity, Na+-ATPase activity, and phosphorylation from ATP i
n mutants Glu781-->Ala and Thr809-->Ala were strongly reduced relative
to the affinity of the wild type (6-8-fold increase in K-0.5 for Nain the phosphorylation assay for both mutants). The Glu781-->Ala mutan
t displayed a 3-4-fold reduction in the apparent affinity for K+ and w
as able to hydrolyze ATP at a high rate in the absence of K+(V-max for
Na+-ATPase activity 5-fold higher than that of the wild-type enzyme).
The steady-state phosphoenzyme level formed by the Glu781-->Ala mutan
t was increased 3-fold by addition of oligomycin, whereas only a sligh
t effect of oligomycin was observed for mutant Thr809-->Ala and the wi
ld type. Using the steady-state phosphoenzyme level determined in the
presence of oligomycin as a measure of the concentration of active enz
yme sites, a maximum molecular turnover number for the Na+,K+-ATPase r
eaction was calculated to be slightly lower for mutant Glu781-->Ala th
an the maximum turnover numbers of the wild type and mutant Thr809-->A
la.