TY - JOUR
T1 - Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line
AU - Ismail-Beigi, F.
AU - Pressley, T. A.
AU - Haber, R. S.
AU - Gick, G. G.
AU - Loeb, J. N.
AU - Edelman, I. S.
PY - 1988
Y1 - 1988
N2 - Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (K(I) = 1 x 10-4 M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ('back-door') phosphorylation, such that the calculated catalytic turnover number of ~8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. ~1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under V(max) conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both nasal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase α-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the α-II and α-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the α-I isoform is expressed under control and low-K+ conditions.
AB - Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (K(I) = 1 x 10-4 M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ('back-door') phosphorylation, such that the calculated catalytic turnover number of ~8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. ~1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under V(max) conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both nasal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase α-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the α-II and α-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the α-I isoform is expressed under control and low-K+ conditions.
UR - http://www.scopus.com/inward/record.url?scp=0023928051&partnerID=8YFLogxK
M3 - Article
C2 - 2836415
AN - SCOPUS:0023928051
SN - 0021-9258
VL - 263
SP - 8162
EP - 8167
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -