Carilli C T, Berne M, Cantley L C, Haupert G T
J Biol Chem. 1985 Jan 25;260(2):1027-31.
We have characterized the effect of a stable small molecule isolated from bovine hypothalamus (Haupert, G. T., and Sancho, J. M. (1979) Proc. Natl. Acad. Sci. 76, 4658-4660) on mammalian (Na,K)ATPase. This hypothalamus-derived inhibitory factor, HIF, has been shown to inhibit ATPase activity of purified dog kidney enzyme reversibly with high affinity (Haupert, G. T., Carilli, C. T., and Cantley, L. C. (1984) Am. J. Physiol. 247, F919-F924). In this report it is shown that HIF inhibits the ouabain sensitive component of 86Rb+ uptake into human red blood cells. HIF also inhibited (Na,K)ATPase activity of unsealed red cell membranes but not that of sealed inside-out vesicles, indicating that HIF is impermeant to red cell membranes and inhibits the (Na,K)ATPase from the extracellular side. In unsealed human red cell membranes, concentrations of HIF which caused 70% inhibition of the (Na,K)ATPase did not inhibit ATP hydrolysis by plasma membrane (Ca2+)ATPase or (Mg2+)ATPase. However, at a similar concentration, HIF was shown to inhibit rabbit muscle sarcoplasmic reticulum (Ca2+)ATPase. HIF also inhibited p-nitrophenylphosphatase activity of unmodified or fluorescein-5'-iso-thiocyanate labeled dog kidney (Na,K)ATPase. As judged by fluorescein fluorescence of the modified enzyme, HIF stabilized the low fluorescent "E2" conformation of the enzyme similar to that stabilized by ouabain. However, unlike ouabain, HIF blocked covalent phosphorylation of dog kidney (Na,K)ATPase by inorganic phosphate. These studies show that HIF is an inhibitor of (Na,K)ATPase which acts from the extracellular side of the membrane by a mechanism similar to but not identical to that of cardiac glycosides.
我们已经对从牛下丘脑分离出的一种稳定小分子（豪珀特，G.T.，和桑乔，J.M.（1979年）《美国国家科学院院刊》76卷，4658 - 4660页）对哺乳动物（钠，钾）ATP酶的作用进行了表征。这种源自下丘脑的抑制因子，即HIF，已被证明能以高亲和力可逆地抑制纯化的犬肾酶的ATP酶活性（豪珀特，G.T.，卡里利，C.T.，和坎特利，L.C.（1984年）《美国生理学杂志》247卷，F919 - F924页）。在本报告中表明，HIF抑制86Rb⁺摄取到人类红细胞中的哇巴因敏感成分。HIF还抑制未封闭红细胞膜的（钠，钾）ATP酶活性，但不抑制内翻小泡的（钠，钾）ATP酶活性，这表明HIF不能透过红细胞膜，且从细胞外侧抑制（钠，钾）ATP酶。在未封闭的人类红细胞膜中，导致（钠，钾）ATP酶70%%抑制的HIF浓度并不抑制质膜（钙）ATP酶或（镁）ATP酶的ATP水解。然而，在相似浓度下，HIF被证明能抑制兔肌肌浆网（钙）ATP酶。HIF还抑制未修饰的或用异硫氰酸荧光素 - 5'-标记的犬肾（钠，钾）ATP酶的对硝基苯磷酸酶活性。通过修饰酶的荧光素荧光判断，HIF稳定了该酶的低荧光“E2”构象，类似于哇巴因所稳定化的构象。然而，与哇巴因不同，HIF阻断无机磷酸对犬肾（钠，钾）ATP酶的共价磷酸化作用。这些研究表明，HIF是一种（钠，钾）ATP酶抑制剂，它通过一种与强心苷类似但不完全相同的机制从膜的细胞外侧起作用。
