CYTOCHEMICAL LOCALIZATION OF 5'-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER
CYTOCHEMICAL LOCALIZATION OF 5'-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER
复制标题
从大鼠肝脏分离的亚细胞组分中 5-核苷酸酶的细胞化学定位
作者：
C. Widnell
建立了5‘-核苷酸酶在分离的、未固定的大鼠肝微粒体中的细胞化学定位方法。膜与腺苷5‘-磷酸(5’-AMP)和硝酸铅孵育，然后用蔗糖密度梯度分离：通过这种方法，释放的磷酸盐全部与膜一起回收。2‘-腺苷2’-磷酸(2‘-AMP)和3’，5‘-环磷酸(3’，5‘-AMP)是纯化的5’-核苷酸酶的竞争性抑制剂，但不是底物，并用来确定细胞化学反应的特异性。细胞化学方法的孵育条件不影响5‘-核苷酸酶的特异性。与Pb2+、Pb2+和2‘-AMP或3’，5‘-AMP孵育的对照组微体密度相同，但略高于不加Pb2+孵育的微体，在电子显微镜下观察与铅沉淀无关；Pb2+和5’-AMP孵育的微体密度较高，在电子显微镜下可见磷酸铅染色。Pb2+和无机磷酸盐人工生成的沉淀物与反应产物不同。因此，微体在蔗糖梯度上分离，并对亚组分进行细胞化学检查。铅析出物与大多数表面粗糙的小泡伴生，反应产物在各组分中分布不均。在最浅的亚组分中，可观察到在分离的质膜中可见类似胆小管细胞膜的小泡。反应产物定位于微粒体膜的外表面，并被低浓度的乙二胺四乙酸增溶。结果表明，5‘-核苷酸酶存在于内质网中，微体部分除含有来自质膜的小泡外，还含有小泡。
A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO3)2 and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb2+, or Pb2+ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb2+, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb2+ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb2+ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.