title: Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line.
abstract: 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 (KI = 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 approximately 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. approximately 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 Vmax conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both basal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase alpha-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the alpha-II and alpha-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 alpha-I isoform is expressed under control and low-K+ conditions.
authors.affiliation: Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
authors.forename: F
authors.initials: F
authors.lastname: Ismail-Beigi
authors.forename: T A
authors.initials: TA
authors.lastname: Pressley
authors.forename: R S
authors.initials: RS
authors.lastname: Haber
authors.forename: G G
authors.initials: GG
authors.lastname: Gick
authors.forename: J N
authors.initials: JN
authors.lastname: Loeb
authors.forename: I S
authors.initials: IS
authors.lastname: Edelman
mesh_terms: D000818:Animals; D002460:Cell Line; D003840:Deoxycholic Acid / Q000494:pharmacology; D004790:Enzyme Induction; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis*
chemical_list: D012333:RNA, Messenger; D003840:Deoxycholic Acid; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium