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Homogeneous trinder-coupled assay for the determination of glucose-6-phosphatase activity in tissue extracts

✍ Scribed by Walid G. Yasmineh; Janelle I. Caspers; Athanasios Theologides

Publisher: Elsevier Science
Year: 1992
Tongue: English
Weight: 616 KB
Volume: 25
Category: Article
ISSN: 0009-9120
DOI: 10.1016/0009-9120(92)80053-j

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✦ Synopsis

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%.

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