α亚基氨基末端结构域对Na+/K(+)-ATP酶磷酸中间体的自身调节。

Autoregulation of the phosphointermediate of Na+/K(+)-ATPase by the amino-terminal domain of the alpha-subunit.

作者信息

Huang W H, Ganjeizadeh M, Wang Y H, Chiu I N, Askari A

机构信息

Department of Pharmacology, Medical College of Ohio, Toledo 43699-0008.

出版信息

Biochim Biophys Acta. 1990 Nov 30;1030(1):65-72. doi: 10.1016/0005-2736(90)90239-k.

DOI:10.1016/0005-2736(90)90239-k

PMID:2176103

Abstract

Chymotryptic cleavage of the alpha-subunit of the canine kidney Na+/K(+)-ATPase in the presence of Na+ abolishes ATPase activity and yields an 83 kDa peptide from Ala 267 to the COOH-terminus. To test the proposal that E1 to E2 conformational transition is blocked in this modified enzyme, we have made a detailed comparison of its phosphorylation with that of the native enzyme by ATP. While phosphorylation of alpha is dependent on Na+ and prevented by K+, that of the 83 kDa peptide is modestly stimulated by Na+; and only this stimulation, but not the Na(+)-independent phosphorylation is inhibited by K+. Ouabain, which inhibits alpha-phosphorylation by ATP, activates Na(+)-independent phosphorylation of the 83 kDa peptide by ATP, and inhibits the Na(+)-stimulation of this process. While there is a ouabain-stimulated phosphorylation of alpha by Pi, the 83 kDa peptide is not phosphorylated by Pi with or without ouabain. In its sensitivity to ADP, and insensitivity to K+, the phosphopeptide is similar to the E1P of the native enzyme; however, the spontaneous decomposition rate of the phosphopeptide is orders of magnitude lower than that of the native EP. Na+ has no effect on the spontaneous decomposition of the phosphopeptide; but at high Na+ concentrations (K0.5 = 350 mM) the ADP sensitivity of the phosphopeptide is reduced. The phosphopeptide, like the native EP, is acid-stable, alkaline-labile, and sensitive to hydroxylamine and molybdate. The chymotrypsin-treated enzyme catalyzes an ADP-ATP exchange activity that is stimulated by Na+. The Na(+)-independent part of this exchange, unlike that of the native enzyme, is activated by ouabain. Our findings establish that (a) the phosphorylation process and its control by Na+, K+ and ouabain are autoregulated by the NH2-terminal domain of the alpha-subunit; and (b) the often repeated assumption that the primary role of this domain is in the regulation of E1-E2 transitions is not valid.

摘要

在存在Na⁺的情况下，用胰凝乳蛋白酶切割犬肾Na⁺/K⁺-ATP酶的α亚基会消除ATP酶活性，并产生一个从Ala 267到COOH末端的83 kDa肽段。为了验证在这种修饰酶中E1到E2构象转变受阻的提议，我们对其与天然酶被ATP磷酸化的情况进行了详细比较。虽然α亚基的磷酸化依赖于Na⁺并受K⁺抑制，但83 kDa肽段的磷酸化受到Na⁺适度刺激；并且只有这种刺激，而不是不依赖Na⁺的磷酸化，受到K⁺抑制。哇巴因抑制ATP对α亚基的磷酸化，却激活ATP对83 kDa肽段的不依赖Na⁺的磷酸化，并抑制该过程中Na⁺的刺激作用。虽然存在哇巴因刺激Pi对α亚基的磷酸化，但无论有无哇巴因，Pi都不会使83 kDa肽段磷酸化。在对ADP的敏感性和对K⁺的不敏感性方面，磷酸肽与天然酶的E1P相似；然而，磷酸肽的自发分解速率比天然EP低几个数量级。Na⁺对磷酸肽的自发分解没有影响；但在高Na⁺浓度（K0.5 = 350 mM）下，磷酸肽对ADP的敏感性降低。磷酸肽与天然EP一样，对酸稳定，对碱不稳定，并且对羟胺和钼酸盐敏感。经胰凝乳蛋白酶处理的酶催化一种受Na⁺刺激的ADP-ATP交换活性。与天然酶不同，这种交换的不依赖Na⁺部分被哇巴因激活。我们的发现表明：（a）磷酸化过程及其受Na⁺、K⁺和哇巴因的调控由α亚基的NH2末端结构域自动调节；（b）该结构域的主要作用是调节E1-E2转变这一经常被重复的假设并不成立。