Glucose-6-phosphatase activity, assay

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-... 

However, the acid phosphatase activity of rat iiver lysosomes has recently been resolved into at least two enzymes [531]. Acid phosphatase is used in subcellular fractionation studies as a marker enzyme for lysosomes. Both acid phosphatase [E.C. 3.1.3.2] and alkaline phosphatase [E.C. 3.1.3.1] activities should not be confused with other specific phosphatases with high specificity requirements for substrate, e.g. glucose-6-phos-phatase, fructose-l,6-diphosphatase, phosphatidate phosphatase. Several assay procedures are available, u.v. estimation can be achieved using phosphoenolpyruvate as a substrate and lactate dehydrogenase in an indicator reaction [539]. Colorimetric assays can be based upon the liberation of phenol from phenylphosphate [540], upon the Uberation of phosphate from sodium /3-glycerophosphate [541], upon the hydrolysis of sodium phenolphthalein phosphate [542], or upon the hydrolysis of p-nitrophenyl phosphate [543]. 

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. 

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

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. 

FIGURE 1. Enzyme activities of a soil incubated with glucose and NO3. For phosphatase, proteinase (casein), and proteinase (ZPL) respectively, activities are expressed as [jmoles of p-nitrophenol, tyrosine, and leucine released g soil h For dehydrogenase, activities are expressed in absorbance units xl5 (485 nm) g soil h Conditions of assay have been described. ... 

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. 

---