Wada I, Eto S, Himeno M, Kato K
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
J Biochem. 1987 May;101(5):1077-85. doi: 10.1093/oxfordjournals.jbchem.a121972.
5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.
在纯化的大鼠肝脏微粒体中发现了5'-核苷酸酶。当微粒体被亚分级分离为膜部分和可溶性内容物部分时，发现5'-核苷酸酶总活性的73%存在于膜部分，24%存在于可溶性内容物部分。使用针对大鼠肝脏质膜5'-核苷酸酶的特异性多克隆抗体进行免疫印迹分析表明，膜部分和内容物部分中的两种5'-核苷酸酶在SDS-聚丙烯酰胺凝胶电泳上的迁移率与质膜中的酶相同（Mr = 72,000）。膜部分和内容物部分中的5'-核苷酸酶对神经氨酸酶敏感，消化后转化为分子量小4 kDa的形式，这与质膜酶的情况相同。膜部分和内容物部分的5'-核苷酸酶均使用免疫亲和色谱法进行纯化，并对纯化酶的等电点、热稳定性和寡聚结构进行了比较。等电聚焦和热稳定性测试表明可溶性酶与膜结合酶相似。然而，膜结合酶在没有Triton X-100的情况下会聚集，而可溶性酶表现为二聚体。使用针对5'-核苷酸酶的抗体和神经氨酸酶处理研究了微粒体膜中5'-核苷酸酶的拓扑结构。在完整的微粒体部分中未观察到5'-核苷酸酶的抑制作用，直到微粒体因渗透休克而被破坏。这些结果表明5'-核苷酸酶的活性位点和寡糖链位于微粒体膜的内表面。
