CYTOCHEMICAL LOCALIZATION OF 5'-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER

作者：Christopher C. Widnell · 发表于：The Journal of Cell Biology · 年份：1972 · DOI：10.1083/jcb.52.3.542 · 被引用次数：191 · 研究领域：Adenosine and Purinergic Signaling、Neonatal Health and Biochemistry、Infant Nutrition and Health

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(NO(3))(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 Pb(2+), or Pb(2+) and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb(2+), and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb(2+) and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb(2+) 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 ...