PMID- 31295070
OWN - NLM
STAT- MEDLINE
DCOM- 20201009
LR  - 20201009
IS  - 1522-1601 (Electronic)
IS  - 0161-7567 (Linking)
VI  - 127
IP  - 3
DP  - 2019 Sep 1
TI  - Rapid radiochemical filter paper assay for determination of hexokinase activity 
      and affinity for glucose-6-phosphate.
PG  - 661-667
LID - 10.1152/japplphysiol.00207.2018 [doi]
AB  - 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 a 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 K(M). 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.NEW & NOTEWORTHY Traditionally, hexokinase 
      activity has been analyzed spectrophotometrically in which the product formation 
      of glucose-6-phosphate (G6P) is analyzed by an indirect reaction coupled to NADPH 
      formation during conversion of G6P to 6-P gluconolactone. By nature, this assay 
      prevents measurements of hexokinase (HK) affinity for inhibition by G6P. We have 
      developed a rapid radiochemical filter paper assay to study HK affinity for G6P 
      by use of radiolabeled 2-deoxyglucose as substrate to study physiological 
      regulation of HK affinity for G6P-induced inhibition.
FAU - Hingst, Janne R
AU  - Hingst JR
AD  - Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, 
      Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
FAU - Bjerre, Rie D
AU  - Bjerre RD
AD  - Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, 
      Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
FAU - Wojtaszewski, Jorgen F P
AU  - Wojtaszewski JFP
AD  - Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, 
      Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
FAU - Jensen, Jorgen
AU  - Jensen J
AD  - Department of Physical Performance, Norwegian School of Sports Sciences, Oslo, 
      Norway.
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20190711
PL  - United States
TA  - J Appl Physiol (1985)
JT  - Journal of applied physiology (Bethesda, Md. : 1985)
JID - 8502536
RN  - 3573-50-0 (2-deoxyglucose-6-phosphate)
RN  - 56-73-5 (Glucose-6-Phosphate)
RN  - EC 2.7.1.1 (Hexokinase)
SB  - IM
MH  - Animals
MH  - Glucose-6-Phosphate/analogs & derivatives
MH  - Hexokinase/*analysis/antagonists & inhibitors/metabolism
MH  - Male
MH  - Physical Conditioning, Animal/*physiology
MH  - Radiochemistry/*methods
MH  - Rats, Wistar
OTO - NOTNLM
OT  - hexokinase
EDAT- 2019/07/12 06:00
MHDA- 2020/10/10 06:00
CRDT- 2019/07/12 06:00
PHST- 2019/07/12 06:00 [pubmed]
PHST- 2020/10/10 06:00 [medline]
PHST- 2019/07/12 06:00 [entrez]
AID - 10.1152/japplphysiol.00207.2018 [doi]
PST - ppublish
SO  - J Appl Physiol (1985). 2019 Sep 1;127(3):661-667. doi: 
      10.1152/japplphysiol.00207.2018. Epub 2019 Jul 11.