A competitive binding assay for fructose 2,6-bisphosphate

Helmut Thomas, Kosaku Uyeda

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol protomer) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6-[2-32P]P2 and samples containing varying concentrations of fructose 2,6-P2. The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P2, ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. This new assay is simple, direct, rapid, and does not require pretreatment of tissue extracts.

Original languageEnglish (US)
Pages (from-to)50-56
Number of pages7
JournalAnalytical Biochemistry
Volume154
Issue number1
DOIs
Publication statusPublished - 1986

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Keywords

  • effectors
  • fructose 2,6-P
  • glycolysis
  • phosphofructokinase
  • regulation
  • sugar phosphate

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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