Glucose-6-phosphatase assay methods

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

In the indirect method, crystalline phosphorylase a is incubated with the enzyme preparation to be assayed, aliquots are withdrawn, diluted (which stops the phosphatase reaction), and the diluted solutions are assayed in the absence of 5 -AMP for remaining phosphorylase a activity by incubation with glucose-l-phosphate and glycogen and determining the inorganic phosphate released. 

The hexokinase for use in ATP determination by this method can be obtained from baker s yeast using the isolation procedure of Berger et al. (1) or of Meyerhof (14). Crystalline hexokinase is unnecessary but the preparation should be free of ATPase and other phosphatase activity. (Partially purified hexokinase is now available commercially from Pabst Laboratories.) An aliquot of the extract to be tested is assayed for total 7-minute hydrolyzable phosphate (8). Another aliquot is incubated with hexokinase plus glucose, and the 7-minute hydrolyzable phosphate is again determined. Inasmuch as the glucose-6-phosphate produced by the action of hexokinase on ATP and glucose is not acid labile, the decrease in acid-labile phosphorus is an index of the amount of ATP present. 

L. Hue and H,G. Hers recently described a method employing double labelled glucose (2 - and U-C ) to assay for the activity of liver glucose-6-phosphatase in vivo. The test is based on the principle that, a differential in the label of as compared to glucose is produced during the recycling of glucose at the level of the phosphohexose-isomerase reaction. 

---