Hypothalamic Na(+)-K(+)-ATPase inhibitor characterized in two-sided liposomes containing pure renal Na(+)-K(+)-ATPase.
Hypothalamic Na(+)-K(+)-ATPase inhibitor characterized in two-sided liposomes containing pure renal Na(+)-K(+)-ATPase.
复制标题
下丘脑 Na( )-K( )-ATP 酶抑制剂，其特征在于含有纯肾 Na( )-K( )-ATP 酶的两侧脂质体。
DOI：
10.1152/ajprenal.1990.258.1.f144
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
HaupertJr,GT
中科院分区：
文献类型：
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作者：
Anner,BM;Rey,HG;Moosmayer,M;Meszoely,I;HaupertJr,GT
Na(+)-K(+)-ATP酶的推定内源性抑制剂的功能表征受到从生物来源中提取的少量物质的极大阻碍。因此，我们使用了一种小型化的两侧测试系统，该系统由 ATP 填充的脂质体组成，其中含有分散的、随机定向的肾 Na(+)-K(+)-ATP 酶分子，以研究从牛下丘脑提取的低分子量、非肽 Na(+)-K(+)-ATP 酶抑制剂的作用。利用该测试系统，可以测量单剂量0.1 U（相当于75 fmol）下丘脑抑制因子（HIF）产生的Na（+）-K（+）-ATP酶抑制以及单个单位（约等于750 fmol）的膜渗透性，并且可以估计每单位HIF分子的最小数量。通过正协同性的分子机制，HIF 有效且完全阻断由右侧向外定向的泵群催化的活性 86Rb+ 转运，平均 50% 抑制浓度为 3.5 x 10(-8) M，表明其表观亲和力比哇巴因高约 30 倍。通过研究对由内向外的泵群的抑制，将 HIF 的膜渗透性与已知疏水性的各种强心苷的膜渗透性进行比较，进一步表明，HIF 并不完全像哇巴因，因为 HIF 可以穿透脂质体膜，而哇巴因则不能。除了强心苷之外，HIF 是迄今为止在纯化系统中测试的唯一一种表现出如此惊人的转运抑制作用的化合物。其他已知或提出的内源性Na(+)-K(+)-ATP酶抑制剂，包括不饱和脂肪酸、海藻毒素、脱氢表雄酮和钒酸盐，即使在高浓度下也仅产生部分转运抑制。
The functional characterization of putative endogenous inhibitors of the Na(+)-K(+)-ATPase has been greatly hindered by spare amounts extractable from biological sources. We therefore used a miniaturized, two-sided test system consisting of ATP-filled liposomes containing dispersed, randomly oriented renal Na(+)-K(+)-ATPase molecules to study effects of a low-molecular-weight, nonpeptidic Na(+)-K(+)-ATPase inhibitor extracted from bovine hypothalamus. With this test system, Na(+)-K(+)-ATPase inhibition produced by a single dose of 0.1 U (congruent to 75 fmol) of the hypothalamic inhibitory factor (HIF) as well as the membrane permeation of a single unit (approximately equal to 750 fmol) became measurable, and an estimation of the minimal number of HIF molecules per unit could be made. By a molecular mechanism involving positive cooperativity, HIF potently and completely blocked active 86Rb+ transport catalyzed by the right-side-out-oriented pump population, with an average 50% inhibitory concentration of 3.5 x 10(-8) M, indicating a roughly 30-fold higher apparent affinity than ouabain. By studying inhibition of the inside-out-oriented pump population, comparison of the membrane permeability of HIF to that of various cardiac glycosides of known hydrophobicity further indicated that HIF is not entirely ouabain-like as HIF penetrates the liposomal membrane, whereas ouabain does not. Besides the cardiac glycosides, HIF is the only compound tested thus far in the purified system that displays such striking transport inhibition. Other known or proposed endogenous Na(+)-K(+)-ATPase inhibitors, including unsaturated fatty acids, palytoxin, dehydroepiandrosterone, and vanadate, produce only partial transport inhibition even at high concentration.