Hansen O
Department of Physiology, Aarhus University, Ole Worms Allé 160, DK-8000 Arhus C, Denmark.
Pflugers Arch. 1999 Mar;437(4):517-22. doi: 10.1007/pl00008089.
Purified Na+/K+-ATPase (EC 3.6.1.37) isolated from the rectal gland of Squalus acanthias was characterized in ouabain-binding studies and with respect to isoform(s) of the alpha peptide. To avoid enzyme inactivation [3H]ouabain equilibrium binding was carried out at 20 degrees C. The heterogeneity of Na+/K+-ATPase isolated from shark rectal gland was similar in [3H]ouabain binding as previously seen in hydrolytic studies. The binding isotherms were compatible with the existence of a high-affinity (Kdis 0.69 nM) and a low-affinity (Kdis 42 nM) component of 1.46 and 0.79 nmol.(mg protein)-1, respectively. In Western blots the alpha peptide of the enzyme hybridized with an isoform-specific polyclonal antibody raised to an alpha3-specific region of the large intracellular domain of rat Na+/K+-ATPase, but not with the supposed alpha3-specific monoclonal antibody MA3-915 with its epitope near the N-terminus. Semi-quantitative analysis of the reaction of the alpha3-specific polyclonal antibody with the alpha peptide from the shark enzyme compared to the reaction with alpha peptide from rat brain enzyme indicated that this region is not exactly the same in the two species. The alpha peptide of shark enzyme was not recognized by alpha1- or alpha2-specific polyclonal antibodies, or by the alpha1-specific monoclonal antibodies 3B and F6. The large intracellular domain of Na+/K+-ATPase from shark rectal gland thus seems to be alpha3-like and no alpha isoform heterogeneity seems able to account for the heterogeneity seen in ouabain binding.
从棘鲛直肠腺中分离出的纯化钠钾ATP酶（EC 3.6.1.37），通过哇巴因结合研究以及α肽亚型进行了表征。为避免酶失活，[3H]哇巴因平衡结合在20℃下进行。从鲨鱼直肠腺分离出的钠钾ATP酶的异质性在[3H]哇巴因结合方面与先前水解研究中所见相似。结合等温线与分别为1.46和0.79 nmol·（mg蛋白质）-1的高亲和力（Kdis 0.69 nM）和低亲和力（Kdis 42 nM）成分的存在相符。在蛋白质免疫印迹中，该酶的α肽与针对大鼠钠钾ATP酶大细胞内结构域的α3特异性区域产生的亚型特异性多克隆抗体杂交，但不与假定的α3特异性单克隆抗体MA3 - 915杂交，其表位靠近N端。与大鼠脑酶α肽的反应相比，对α3特异性多克隆抗体与鲨鱼酶α肽反应的半定量分析表明，这两个物种的该区域并不完全相同。鲨鱼酶的α肽未被α1或α2特异性多克隆抗体或α1特异性单克隆抗体3B和F6识别。因此，鲨鱼直肠腺钠钾ATP酶的大细胞内结构域似乎类似α3，并且似乎没有α亚型异质性能够解释在哇巴因结合中看到的异质性。
