Homogeneous trinder-coupled assay for the determination of glucose-6-phosphatase activity in tissue extracts.
We describe an automated, homogeneous, glucose oxidase-coupled method for the determination of glucose-6-phosphatase activity in tissue extracts. The method is based on measurement of the rate of glucose formation by the Trinder reaction, in which the end product is a quinoneimine dye which absorbs maximally at 505 nm and has a molar extinction coefficient of 5700. The incubation mixture contains 20 microL of tissue extract, 25 microL of 0.5 M phosphate buffer, pH 7.0, 175 microL of Trinder/glucose-6-phosphate reagent, and 30 microL of distilled water. After a delay period of 15 min, to exhaust any glucose endogenously present in the extract, glucose production from glucose-6-phosphate is monitored at 505 nm for 5 min in a centrifugal analyzer. The Km was 13 mM over a 10-fold range in glucose-6-phosphate concentration and the reaction was linear up to about 250 U/L. Within-run CV of the assay at activities of 48 and 190 U/L ranged between 2.5-5.0%. The between-run CV at 190 U/L was 5.1%.
Yasmineh
W G
WG
Department of Laboratory Medicine and Pathology, University Hospital, Minneapolis, MN 55455.
Caspers
J I
JI
Theologides
A
A
D000818:Animals; D001322:Autoanalysis; D005947:Glucose / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000032:analysis*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051381:Rats
D005952:Glucose-6-Phosphatase; D005947:Glucose