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 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.
Broschat
Kay O
KO
Cardiovascular and Metabolic Diseases, Pharmacia Corp., 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA.
Gorka
Christine
C
Kasten
Thomas P
TP
Gulve
Eric A
EA
Kilpatrick
Brian
B
D000818:Animals; D002460:Cell Line; D002855:Chromatography, Thin Layer / Q000379:methods; D004795:Enzyme Stability; D005636:Fructosephosphates / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D011874:Radiometry / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics
D005636:Fructosephosphates; D011994:Recombinant Proteins; D005973:Glutamine; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine