A Radiometric Assay for Glutamine:fructose-6-phosphate Amidotransferase

Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first step in the biosynthesis of amino sugars by transferring the amino group from l-glutamine to the acceptor substrate, fructose 6-phosphate, generating the products glucosamine 6-phosphate and glutamic acid. We describe a metho...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2002-06, Vol.305 (1), p.10-15
Main Authors: Broschat, Kay O., Gorka, Christine, Kasten, Thomas P., Gulve, Eric A., Kilpatrick, Brian
Format: Article
Language:English
Subjects:
Animals
Cell Line
Chromatography, Thin Layer - methods
Enzyme Stability
Fructosephosphates - analysis
Fructosephosphates - biosynthesis
Fructosephosphates - metabolism
Glucosamine - analogs & derivatives
Glucosamine - analysis
Glucosamine - biosynthesis
Glucosamine - chemistry
Glucose-6-Phosphate - analogs & derivatives
Glucose-6-Phosphate - analysis
Glucose-6-Phosphate - biosynthesis
Glucose-6-Phosphate - chemistry
Glucose-6-Phosphate - metabolism
Glutamine - analysis
Glutamine - chemistry
Glutamine - metabolism
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) - analysis
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) - antagonists & inhibitors
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) - metabolism
Humans
Kinetics
Linear Models
Radiometry - methods
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Reproducibility of Results
Sensitivity and Specificity
Spodoptera - enzymology
Spodoptera - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first step in the biosynthesis of amino sugars by transferring the amino group from l-glutamine to the acceptor substrate, fructose 6-phosphate, generating the products glucosamine 6-phosphate and glutamic acid. We describe a method for the synthesis and purification of the substrate, fructose 6-phosphate, and methods for a radiometric assay of human GFAT1 that can be performed in either of two formats: a small disposable-column format and a high-throughput 96-well-plate format. The method performed in the column format can detect 1 pmol of glucosamine 6-phosphate, much less than that required by previously published assays that measure GlcN 6-phosphate. The column assay demonstrates a broad linear range with low variability. In both formats, the assay is linear with time and enzyme concentration and is highly reproducible. This method greatly improves the sensitivity and speed with which GFAT1 activity can be measured and facilitates direct kinetic measurement of the transferase activity.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2002.5625