Baron M D, Pope B, Luzio J P
Biochem J. 1986 Jun 1;236(2):495-502. doi: 10.1042/bj2360495.
The transmembrane topography of the rat hepatocyte ectoenzyme 5'-nucleotidase was studied by the use of glycoprotein labelling and limited-proteolysis techniques. Comparison, by one-dimensional peptide mapping, of enzyme iodinated from outside the cell with that iodinated in the solubilized state showed that no additional iodination sites were revealed on solubilization. Incubation of newly synthesized enzyme in a microsomal membrane fraction with proteinase showed that the entire molecule of 5'-nucleotidase was protected from proteolysis. These data suggest that little, if any, of the 5'-nucleotidase molecule is present on the cytoplasmic side of the plasma membrane. No evidence was found for a previously proposed interaction between 5'-nucleotidase and actin, although the ability of preparations of 5'-nucleotidase to prevent inhibition of deoxyribonuclease I by actin was explained by minute traces of ATPase activity. Comparison of peptide maps of enzyme labelled by iodination or by methods specific for carbohydrate showed that in both cases predominantly one section of the molecule was labelled. It is proposed that the enzyme is a short-stalked integral membrane protein without a cytoplasmic domain in which about one-third of the molecule forms the accessible molecular surface.
利用糖蛋白标记和有限蛋白酶解技术研究了大鼠肝细胞外切酶5'-核苷酸酶的跨膜拓扑结构。通过一维肽图比较细胞外碘化的酶与溶解状态下碘化的酶，结果表明溶解后未发现额外的碘化位点。将新合成的酶与蛋白酶在微粒体膜组分中孵育，结果表明5'-核苷酸酶的整个分子受到蛋白酶解的保护。这些数据表明，5'-核苷酸酶分子在质膜细胞质侧的存在极少（如果有的话）。尽管5'-核苷酸酶制剂能够防止肌动蛋白对脱氧核糖核酸酶I的抑制作用可由微量的ATP酶活性来解释，但未发现有证据支持先前提出的5'-核苷酸酶与肌动蛋白之间的相互作用。对通过碘化或碳水化合物特异性方法标记的酶的肽图进行比较，结果表明在这两种情况下，分子的主要一个区段都被标记。有人提出该酶是一种短柄整合膜蛋白，没有细胞质结构域，其中约三分之一的分子形成可及的分子表面。
