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J Biol Chem. 1996 Sep 27;271(39):23683-9.

Structure/function analysis of the amino-terminal region of the 1 and 2 subunits of Na,K-ATPase.

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  • 1Department of Medicine, McGill University, Montreal, Canada.


The alpha2 isoform of the Na,K-ATPase exhibits kinetic behavior distinct from that of the alpha1 isoform. The distinctive behavior is apparent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K+ deocclusion pathway of the reaction cycle is rate-limiting; the alpha1 activity is inhibited by K+, whereas alpha2 is stimulated. When 32 NH2-terminal amino acid residues are removed from alpha1, the kinetic behavior of the mutant enzyme (alpha1M32) is similar to that of alpha2 (Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948). In the current study, the region of the alpha1 NH2 terminus involved in modulating this kinetic behavior has been localized to the highly charged sequence comprising residues 24-32. Within this nonapeptide, differences between alpha1 and alpha2 are conservative and are confined to residues 25-27. The behavior of two chimeric enzymes: (i) alpha1 with the first 32 residues identical to the alpha2 sequence, alpha1 (1-32alpha2), and (ii) alpha2 with the first 32 residues identical to the alpha1 sequence, alpha2(1-32alpha1), indicates that the distinctive kinetic behavior of alpha1 and alpha2 is not due to the 24-32 NH2-terminal domain, per se, but rather to its interaction with other, isoform-specific region(s) of the alpha1 protein. We also demonstrate that the distinct K+ activation profiles of either alpha2 or alpha1M32, compared to alpha1 is due to a faster release of K+ from the K+-occluded enzyme, and to a higher affinity for ATP. This was determined in studies using two approaches: (i) kinetic analysis of the reaction modeled according to a branched pathway of K+ deocclusion through low and high affinity ATP pathways and, (ii) measurements of the (rapid) phosphorylation of the enzyme (E1 conformation) by [gamma-32P]ATP following the rate-limiting formation of the K+-free enzyme from the K+-occluded state (E2(K) --> E1 + K+). The observed kinetic differences between alpha2 and alpha1 suggest that these Na,K-ATPase isoforms differ in the steady-state distribution of E1 and E2 conformational states.

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