A rapid radiochemical filter paper assay for determination of Hexokinase activity and affinity for glucose-6-phosphate (G6P)
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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-6-phosphate (2-DG-6P) does not inhibit HK activity. By this we have developed a new method for rapid radiochemical analysis of HK activity with 2-deoxyglucose (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 Vmax and KM. This is the first simple assay evaluating 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.
|Journal||Journal of Applied Physiology|
|Publication status||Accepted/In press - 11 Jul 2019|