Tymiak A A, Norman J A, Bolgar M, DiDonato G C, Lee H, Parker W L, Lo L C, Berova N, Nakanishi K, Haber E
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543.
Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8189-93. doi: 10.1073/pnas.90.17.8189.
Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K(+)-ATPase in humans. We have purified a bovine hypothalamic Na+/K(+)-ATPase inhibitory factor (HIF) by using affinity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.
最近的报告显示，人体内存在一种哇巴因样的Na⁺/K⁺-ATP酶抑制剂。我们通过亲和色谱结合高效液相色谱法纯化了一种牛下丘脑Na⁺/K⁺-ATP酶抑制因子（HIF）。通过离子喷雾质谱法测定，这种抑制剂的分子量为584，与哇巴因等压。通过手性气相色谱/质谱检测，糖苷酶处理或HIF的酸水解仅释放出L-鼠李糖，这是在哇巴因中发现的己糖异构体。此外，酶促生成的去鼠李糖基HIF的分子量为438，与哇巴因的苷元哇巴因苷元相同。通过反相高效液相色谱保留时间，HIF及其苷元分别与哇巴因和哇巴因苷元无法区分。然而，用萘甲酰咪唑衍生化后再进行高效液相色谱分析，发现HIF和哇巴因的萘甲酰化产物保留时间不同。随后对HIF和哇巴因分离的萘甲酰化产物进行圆二色光谱分析，证实它们是不同的。这项研究提供了色谱和光谱证据，表明哇巴因和HIF是同分异构的强心苷。结构差异被认为是HIF和哇巴因在生物学特性上观察到的显著差异的原因。
