Tal D M, Yanuck M D, van Hall G, Karlish S J
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Biochim Biophys Acta. 1989 Oct 2;985(1):55-9. doi: 10.1016/0005-2736(89)90103-x.
A preparative purification of endogenous inhibitors of the Na+/K+-ATPase has been carried out from bovine blood. Dried plasma was deproteinized, hexane-extracted and desalted, followed by further purification through a series of reverse-phase HPLC fractionations. Fractions active in inhibiting Na+/K+-ATPase activity and displacing ouabain were collected and purified further. By comparison with ouabain, the final extract was found to have a steeper concentration-effect curve in the inhibition of Na+/K+-ATPase. In displacement of [3H]ouabain, the extract had again a steeper concentration-effect curve than does ouabain, and in addition it enhanced ouabain binding at high dilutions. These properties are indicative of nonspecific interactions with the Na+/K+-ATPase. The active fraction was identified by TLC, HPLC, NMR, GLC and GC-MS, to be a mixture of three unesterified fatty acids, mainly oleic acid (72% of the total) and three saturated hydrocarbons. The assignment of structures was corroborated by comparison with authentic samples.
已从牛血中对内源性钠钾ATP酶抑制剂进行了制备性纯化。干燥血浆经脱蛋白、己烷萃取和脱盐处理，随后通过一系列反相高效液相色谱分级进一步纯化。收集抑制钠钾ATP酶活性和置换哇巴因的活性级分并进一步纯化。与哇巴因相比，发现最终提取物在抑制钠钾ATP酶方面具有更陡的浓度效应曲线。在置换[3H]哇巴因时，提取物的浓度效应曲线再次比哇巴因更陡，此外，在高稀释度下它还增强了哇巴因的结合。这些特性表明与钠钾ATP酶存在非特异性相互作用。通过薄层色谱、高效液相色谱、核磁共振、气相色谱和气相色谱-质谱鉴定活性级分为三种未酯化脂肪酸的混合物，主要是油酸（占总量的72%）和三种饱和烃。通过与标准样品比较证实了结构归属。
