3-Glucuronidase assay

F4. Fishman, W. H., Springer, B., and Brunetti, 11., Application of an improved glucuronidase assay method to the study of human blood /3-glucuronidase. J. Biol. Chem. 173, 449-456 (1948). 

With phenolphthalein j3-D-glucopyranosyluronic acid as substrate in a human serum jS-D-glucuronidase assay, centrifugation at high speed after addition of alkali was required to minimize blanks.Optimal substrate concentration was 3-6 mM excess substrate was inhibitory. 

The activity of j8-glucuronidase at the usual pH of the transferase assays (pH 7.4r-8.2) is very low. The enzyme, most likely, has no role in the conjugation process itself (G4). If potentially important, e.g., in work with homogenates or cell extracts containing partially lysed lysosomes, the specific inhibitor saccharo-(l4)-lactone (L9) can be added to the incubation mixtures. 

Groves and Teng (1992) investigated the effect of compactional pressure on biologically active proteinaceous enzymes such as a-amylase, P-glucuronidase, lipase, and urease. Assaying the activity of these enzymes before and after the compaction... 

To overcome this problem, an agar diffusion test has been developed to detect chloramphenicol residues in the urine of slaughtered animals (87). The principle of the test is based upon incorporation of -glucuronidase in an agar medium shown with Bacillus subtilis. With this test, the glucuronide of chloramphenicol, which is the major metabolite in urine, is hydrolyzed and the antibacterial activity is demonstrated according to the usual microbiological detection assays. 

Coumarins also have a C6-C3 skeleton, but they possess an oxygen heterocycle as part of the C3-unit. There are numerous coumarins, many of which play a role in disease and pest resistance, as well as UV-tolerance. The coumarin umbelliferone (1.21) is popular in enzyme assays. Umbelliferone esters can be used as a substrate for non-specific esterase enzyme assays and in fluorescent immunoassays (Jacks and Kircher, 1967). In order to quantify the enzyme activity of the popular reporter gene P-glucuronidase (GUS), plant extracts can be incubated with 4-methylumbelliferyl P-D-glucuronide (4-MUG 1.22), which upon hydrolysis... 

Some preparations of mammalian /3-glucuronidase of high specific activity display a fall in net activity on dilution, an effect that can be prevented by adding a variety of substances (see Section IX, 2), of which albumin is, perhaps, the most satisfactory.49 138 When this phenomenon is encountered, the enzyme is assayed in the presence of an activator (see Fig. 4), and some of the figures quoted above were obtained in this way, as well as the kinetic constants quoted in subsequent Sections. (Failure to observe the dilution phenomenon with one highly-active preparation,80 although the customary activation was seen with substances like albumin, may be explained by the fact that the enzyme had undergone partial inactivation.)... 

It seems feasible that traces of heavy-metal ions may have some bearing upon certain features of the action of mammalian /3-glucuronidase, such as the fall in net activity seen on dilution of highly purified preparations (see Section IV), the inhibitory action of the unknown constituents of urine,185 190 and the pH optimum at pH 3.4 observed by Mills, Paul, and Smith166 but by no other workers (see Section VI). The presence of traces of Cu in the assay mixture would provide a completely satisfactory explanation of the variable effects reported with L-ascorbic acid (see Section IX, 2).196s... 

Application of LC-MS/MS techniques to the analysis of phthalate ester metabolites in urine have also been developed. For example, Blount et al. (2000b) have developed an assay to quantify the monoester metabolites (including MEHP) of eight phthalate diesters in urine, utilizing HPLC coupled with atmospheric pressure chemical ionization and tandem mass spectrometric (APCI-MS/MS) detection techniques. Urine samples were treated with -glucuronidase to release the free phthalate monoesters followed by a two-step solid phase extraction procedure. After evaporative concentration of the eluant, the analytes in the purified samples are further separated on a phenyl reverse phase HPLC column and quantified by APCI-MS/MS, following careful optizimation of the APCI-MS/MS instrument. The limits of detection for MEHP were determined to be 1.2 ng/ml urine with recovery efficiencies of between 78 and 91%. 

Fig. 7. Changes in the contents of specific mRNAs and nuclear receptors in mouse kidney after exposure to testosterone. Female NCS mice were given a single intraperitoneal dose of 10 mg of testosterone at time zero. Thereafter, nuclear androgen receptors were measured, The specific mRNAs of three proteins were also assayed, namely for ornithine decarboxylase (- -), KAP (-0-) and /3-glucuronidase (- -). Redrawn from Catterall et al. [26],...

Spectrophotometric methods are, of course, not restricted to assays based on the NAD(P)+/NAD(P)H pair of coenzymes, and there are many natural and synthetic substrates whose reactions can be assayed in this way. The p-nitrophenyl group forms the basis of many convenient spectroscopic assays, for example for the enzyme glucuronidase ... 

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