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

Broschat, Kay O.; Gorka, Christine; Kasten, Tom; Gulve, T. Eric A.; Kilpatrick, Brian F.

doi:10.1006/abio.2002.5625

Cited by 9 publications

(6 citation statements)

References 13 publications

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“…However, because of their relative insensitivity, both the Morgan Elson and Glu dehydrogenase methods require appreciable GlcN-6-P accumulation. In this study we report the kinetic characterization of hGFAT1 using a sensitive method similar to that reported by Callahan et al (29), which allows the measurement of GFAT activity with less than 0.2 M product accumulation (30). Our results indicate that hGFAT1 binds Fru-6-P with much greater affinity than previously realized.…”

supporting

confidence: 81%

“…The K m for Fru-6-P measured with overexpressed mouse GFAT1 and GFAT1Alt also differs significantly from that of hGFAT1 in this report (12). Although species or methodological differences might affect these measurements, it is also possible that the presence of phosphoglucoisomerase activity in cytosolic extracts can rapidly deplete Fru-6-P (30). Direct comparison of the biochemical properties of purified GFAT isoforms should be undertaken to better understand the regulation of this important enzyme.…”

Section: Discussionmentioning

confidence: 57%

“…The custom synthesis of [U- 14 C]Fru-6-P (300 mCi/mmol) was performed by American Radiolabeled Chemicals, Inc., St. Louis, MO, according to the methods described elsewhere (30). L-[…”

Section: Methodsmentioning

confidence: 99%

“…Determination of GlcN-6-P Production-The direct measure of [U- 14 C]GlcN-6-P formed from [U- 14 C]Fru-6-P by hGFAT1 was determined by an ion exchange method, which separates the product and substrate on small disposable columns (30). Assay of hGFAT1 activity was measured at room temperature for initial rate measurements.…”

Section: Methodsmentioning

confidence: 99%

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Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Broschat¹,

Gorka²,

Page³

et al. 2002

Journal of Biological Chemistry

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Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K m 0.2-1 mM). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K m for Fru-6-P of 7 M, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K d of 2 M was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K i measured of 6 M. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDPGlcNAc, was competitive with a K i of 4 M. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.

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supporting

confidence: 81%

Section: Discussionmentioning

confidence: 57%

“…The custom synthesis of [U- 14 C]Fru-6-P (300 mCi/mmol) was performed by American Radiolabeled Chemicals, Inc., St. Louis, MO, according to the methods described elsewhere (30). L-[…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Broschat¹,

Gorka²,

Page³

et al. 2002

Journal of Biological Chemistry

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“…Its quantification has been reported using colorimetric Morgan‐Elson assay,16, 17 radiometric assay with [ 14 C]‐fructose‐6P as substrate,24, 25 or HPLC‐based assay after o ‐phthaldialdehyde derivatization 26. Each of these methods has limitations due to interferences with other compounds18–23 or because of their high cost. They are time‐consuming and difficult to adapt to a parallel screening.…”

mentioning

confidence: 99%

Monitoring enzyme‐catalyzed production of glucosamine‐6P by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: a new enzymatic assay for glucosamine‐6P synthase

Maillard¹,

Guérineau²,

Badet‐Denisot³

et al. 2006

Rapid Comm Mass Spectrometry

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A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) method for quantification of D-glucosamine-6P (GlcN-6P) that allows the kinetic study of glucosamine-6P synthase (Glms) is presented. The present report describes the optimization of the different steps of a new enzymatic assay for Glms based on in situ N-acetylation of GlcN-6P and MALDI-TOFMS analysis using N-(13C2)acetylglucosamine-6P as internal standard. Since no isotopically substituted GlcN-6P was available, the N-(13C2)acetyl derivative, easily obtained from (13C4)-acetic anhydride, was used as internal standard. Validation of the assay was achieved by measuring the fructose-6P Michaelis constant, in full agreement with reported values, and by studying the inhibition properties of arabinose-5P oxime.

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Assays for Insulin and Insulin-Like Signal Transduction Based on Adipocytes, Hepatocytes and Myocytes

Müller¹

2016

Drug Discovery and Evaluation: Pharmacological Assays

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

Cited by 9 publications

References 13 publications

Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Monitoring enzyme‐catalyzed production of glucosamine‐6P by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: a new enzymatic assay for glucosamine‐6P synthase

Assays for Insulin and Insulin-Like Signal Transduction Based on Adipocytes, Hepatocytes and Myocytes

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