A rapid radiochemical filter paper assay for determination of hexokinase activity and affinity for glucose-6-phosphate

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Janne Rasmuss Hingst, Rie Dybboe Bjerre, Jørgen Wojtaszewski, Jørgen Jensen

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions.

OriginalsprogEngelsk
TidsskriftJournal of Applied Physiology
Vol/bind127
Udgave nummer3
Sider (fra-til)661-667
Antal sider7
ISSN8750-7587
DOI
StatusUdgivet - 2019

Bibliografisk note

CURIS 2019 NEXS 306

ID: 224997908