Competitive binding assay for fructose 2,6-bisphosphate
A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P/sub 2/ to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol promoter) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6(2-/sup 32/P)P/sub 2/ and samples containing varying concentrations of fructose 2,6-P/sub 2/. 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-P/sub 2/ in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P/sub 2/, 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. The new assay is simple, direct, rapid, and does not require pretreatment.
- Research Organization:
- Veterans Administration Medical Center, Dallas, TX
- OSTI ID:
- 5533729
- Journal Information:
- Anal. Biochem.; (United States), Vol. 154:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
ACID PHOSPHATES
RADIOENZYMATIC ASSAY
FRUCTOSE
HEXOKINASE
CHEMICAL BONDS
ATP
GLYCOLYSIS
INTERFERING ELEMENTS
LABELLED COMPOUNDS
PHOSPHATES
PHOSPHORUS 32
SENSITIVITY
TISSUE EXTRACTS
TRACER TECHNIQUES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
CARBOHYDRATES
DAYS LIVING RADIOISOTOPES
ENZYMES
HEXOSES
ISOTOPE APPLICATIONS
ISOTOPES
KETONES
LIGHT NUCLEI
MATERIALS
METABOLISM
MONOSACCHARIDES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
RADIOISOTOPES
SACCHARIDES
TRANSFERASES
400103* - Radiometric & Radiochemical Procedures- (-1987)
550201 - Biochemistry- Tracer Techniques