An anion-exchange radioassay for glucose 6-phosphate phosphatase: Use in topological studies with endoplasmic reticulum vesicles

Jeffrey S. Rush, Charles J. Waechter

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.

Original languageEnglish
Pages (from-to)328-333
Number of pages6
JournalAnalytical Biochemistry
Volume206
Issue number2
DOIs
StatePublished - Nov 1 1992

Bibliographical note

Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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