Coppi M V, Guidotti G
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Arch Biochem Biophys. 1997 Oct 15;346(2):312-21. doi: 10.1006/abbi.1997.0332.
The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, alpha and beta, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type alpha2 and alpha2L111R,N122D (alpha2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the alpha2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J. Biol. Chem. 266, 16925-16930, 1991) and were introduced into the alpha2 subunit to distinguish its enzymatic activity from that of the endogenous alpha1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha2RD subunit. Despite the fact that S364P substitution does not block association of the alpha2RD subunit with the endogenous beta1 subunit, it prevents the alpha2 and alpha2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.
钠钾ATP酶是哺乳动物细胞中一种重要的质膜转运蛋白，由α和β两个亚基组成，每个亚基都有几种亚型。我们研究了S364P取代对野生型α2以及α2L111R、N122D（α2RD）亚基的亚细胞定位和酶活性的影响。L111R和N122D取代使α2亚基对抑制剂哇巴因的亲和力降低了约一千倍（E.A.朱厄尔和J.B.林格雷尔，《生物化学杂志》266，16925 - 16930，1991），将其引入α2亚基以区分其与COS - 7细胞内源性α1亚基的酶活性。S364P取代位于ATP结合位点，距离钠钾ATP酶催化循环中发生磷酸化的天冬氨酰残基仅五个残基。这种取代显著降低了与α2RD亚基表达相关的酶活性量。尽管S364P取代并不阻止α2RD亚基与内源性β1亚基的结合，但它阻止了α2和α2RD亚基在质膜中积累，并导致它们在内质网中定位。
