Determination of glucose-6-phosphatase activity using the glucose dehydrogenase-coupled reaction.
We present a method to determine glucose 6-phosphate activity. This assay measures the rate of glucose released in the glucose-6-phosphatase reaction. The glucose is oxidized to beta-D-gluconolactone by glucose dehydrogenase in a coupled reaction that uses NAD(P)+. The determination is rapid, reproducible, and does not require withdrawal, precipitation, centrifugation, or neutralization steps. This method provides a simple resolution to the problem of the nonspecific appearance of Pi, which is especially important in studies of regulation of glucose-6-phosphatase performed in the presence of ATP.
Alegre
M
M
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Ciudad
C J
CJ
Fillat
C
C
Guinovart
J J
JJ
D000818:Animals; D005942:Gluconates; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases; D005952:Glucose-6-Phosphatase / Q000032:analysis*; D007783:Lactones; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP; D010084:Oxidation-Reduction; D051381:Rats
D005942:Gluconates; D007783:Lactones; D009249:NADP; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D005952:Glucose-6-Phosphatase; C010730:beta-glucono-1,5-lactone