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  • Title: Functional coupling of phosphorylation and nucleotide binding sites in the proteolytic fragments of Na+/K(+)-ATPase.
    Author: Zolotarjova N, Periyasamy SM, Huang WH, Askari A.
    Journal: J Biol Chem; 1995 Feb 24; 270(8):3989-95. PubMed ID: 7876146.
    Abstract:
    Cleavage of the alpha-subunit of Na+/K(+)-ATPase by trypsin at Arg438-Ala439 causes enzyme inhibition which has been suggested to be due to altered alignment of phosphorylation site on the 48-kDa N-terminal fragment with nucleotide binding site on the 64-kDa C-terminal fragment. Our aims were to test this hypothesis and to assess the effect of the cleavage on the enzyme's two ATP sites. Na(+)-dependent phosphorylation of the partially cleaved enzyme by ATP showed that K0.5 values of ATP for phosphorylations of intact alpha and 48-kDa peptide were the same (0.4 microM). Unchanged interactions among the residues across the cleavage site were also indicated by data showing that reaction of fluorescein isothiocyanate with the 64-kDa peptide blocked phosphorylation of the 48-kDa peptide by ATP. ATP is known to block the reaction of fluorescein isothiocyanate with the enzyme. Experiments on the partially cleaved enzyme showed that K0.5 of ATP for protection of alpha was 30-60 microM, and the value for the protection of interacting 48-kDa and 64-kDa peptides was 1-3 mM. Evidently, while the cleavage does not affect the high affinity catalytic site, it disrupts the allosteric low affinity ATP site. Experiments on reconstituted preparations showed that the cleavage abolished ATP-dependent Na+/K+ exchange, Pi+ATP-dependent Rb+/Rb+ exchange, ATP-dependent Na+/Na+ exchange, and ADP+ATP-dependent Na+/Na+ exchange activities. Selective disruption of the low affinity ATP site accounts for the inhibitions of all functions involving K+(Rb+), based on the established role of this site in the control of K+ access channels. Cleavage-induced inhibitions of other activities, however, suggest additional roles of the low affinity ATP site in the reaction cycle.
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