3-Glucuronidase specificity

Several extraction techniques have also been described that use enzymatic or chemical reactions to improve extraction efficiency. A technique that has been used to increase the overall recovery of the marker residue is enzymatic hydrolysis to convert specific phase II metabolites (glucuronides or sulfates) back into the parent residue. Cooper etal used a glucuronidase to increase 10-fold the concentration of chloramphenicol residues in incurred tissue. As an example of a chemical reaction, Moghaddam et al. used Raney nickel to reduce thioether bonds between benomyl and polar cellular components, and as a result achieved a substantially improved recovery over conventional solvent extraction. In choosing to use either of these approaches, thorough characterization of the metabolism in the tissue sample must be available. 

Transgenic tomato plants Wound induced vascular bundle-specific expression of P-glucuronidase gene Jacinto et al. (14)... 

Three isoenzymes of carboxylesterase were purified from rat liver micro-somes and were named RL1, RL2, and RH1. These differ from each other in their response to hormone treatment, inducibility, substrate specificity, and immunological properties [75], It was shown that RL1, RL2, and RH1 resemble hydrolases p/ 6.2/6.4, pI 6.0, and pI 5.6, respectively. Enzyme RL2 was found to be identical to egasyn, a protein with esterase activity found in the endoplasmic reticulum [76], The role of egasyn is to stabilize glucuronidase (EC 3.2.1.31) by noncovalent binding to the microsomal membrane. 

While some of these reactions are mediated by enzymes, e.g., glucuronidase, which hydrolyze glucuronide conjugates, many others are nonenzy-matic. These are driven by the electrophilicity of the substrates, which renders them susceptible to attack by the HO- anion (specific base catalyzed hydrolysis) or by other bases (general base catalyzed hydrolysis). A semblance of order in such a heterogeneous group of reactions is, thus, far from obvious. 

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. 

The xylanolytic enzyme system of Trichoderma reesei, a well-known producer of cellulolytic enzymes, is versatile and well suited for the total hydrolysis of different xylans. It consists of two major, specific and several non-specific xylanases, at least one / -xylosidase, a-arabinosidase and a-glucuronidase and at least two acetyl esterases. The hydrolysis of polymeric xylans starts by the action of endoxylanases. The side-groupcleaving enzymes have their highest activities towards soluble, short xylo-oligosaccharides, and make the substituted oligosaccharides again accessible for xylanases and / -xylosidase. 

Enzymes. See also, Amylases, Galactosi-dases, /3-Glucuronidase, Glyco-enzymes, a-D-Mannosidase. acting on pectic substances, 5, 79-102 degradation by, of starch and glycogen, 3, 251-310 17, 407-430 of glycoprotein structure, 27, 301-341 specificity of, in the domain of carbohydrates, 5, 49-78... 

The enzymatic hydrolysis is the method of choice when undesired reactions are to be avoided. /Tglucosidases (or /J-glucuronidases) from Helix pomatia digestive juice are now commercially available. They usually show a good hydrolytic performance, even if sometimes need long time reactions (days). Other enzymes are reported to have been used to specifically hydrolize saccharidic bonds like hesperidinase, /J-xylosidase, j3-galactosidase, and mixed crude enzymes like cellulase. 

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.)... 

The only non-mammalian /8-glucuronidase that has been subjected to systematic purification is the enzyme from sheep-rumen microorganisms. After repeated ammonium sulfate fractionation, Marsh101 obtained a colorless preparation with a specific activity of 1,900 (400-fold purification and 2% recovery). The final product gave a linear, specific-property, solubility test from which a figure of 2,200 was derived for the ultimate specific activity of the enzyme, but it was considered that the enzyme may have formed a solid solution with inactive protein. 

---