Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. A radiochemical method for the studies on the microsomal UDPglucuronic acid metabolism has been developed. 2. The rat liver microsomes caused a rapid hydrolysis of UDPglucuronic acid to D-glucuronic acid 1-phosphate and further although much slower to free D-glucuronic acid. In Tris-HCl buffer (pH 7.4) they were produced in ratio 72 : 1. No other metabolites were found in measurable amounts. The pyrophosphatase splitting UDPglucuronic acid showed a pH optimum at 8.9, but the liberation of D-glucuronic acid from UDPglucuronic acid had two pH maxima (pH 3.5 and 8.5). EDTA appeared to be less powerful inhibitor of pyrophosphatase than previously suggested. About 25 per cent of the UDPglucuronic acid hydrolyzing activity was still remaining in the presence of 10 mM EDTA. D-Glucaro-1,4-lactone was found to have a slight inhibitory action on the pyrophosphatase activity. Citrate inhibited powerfully the hydrolysis of UDPglucuronic acid and the liberation of free D-glucuronic acid. Phosphate was also inhibitory. 3. In the presence of an exogenous UDPglucuronosyltransferase substrate, 4-nitrophenol, the formation of D-glucuronic acid 1-phosphate and free D-glucuronic acid were slightly reduced, and D-glucuronic acid 1-phosphate, 4-nitrophenylglucuronide and free D-glucuronic acid were produced in ratio 78 : 23 : 1. When 10 mM EDTA was added to diminish the hydrolytic consumption of the glucuronyl donor substrate, the corresponding ratio was still as unfavorable as 19 : 2.6 : 1. The measurable activity of UDPglucuronosyltransferase was lower in the presence of phosphate or citrate than in Tris-HCl buffer, although they protected the glucuronyl donor substrate against hydrolysis. 4. The results indicate that even in the presence of added glucuronyl acceptor substrate the hydrolysis of UDPglucuronic acid predominates the conjugation in rat liver microsomes. The rate of the hydrolysis of UDPglucuronic acid is quite considerable even in the presence of EDTA, and it is recommended to control the UDPglucuronic acid pyrophosphatase activity when UDPglucuronosyltransferase and glucuronidation reactions are studied. Free D-glucuronic acid appears to be produced from UDPglucuronic acid for further use via D-glucuronic acid 1-phosphate, the rate-limiting step being the hydrolysis of this intermediate. UDP-glucuronosyltransferase, glucuronides of either endogenous or exogenous aglycones and beta-glucuronidase have only a minor role in this respect in rat liver microsomes.
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PMID:Pyrophosphatase and glucuronosyltransferase in microsomal UDPglucuronic-acid metabolism in the rat liver. 0 Dec 76

Since human colorectal tumors are insensitive to most chemotherapeutic agents, there is a need for the discovery of new drugs that would show activity against this disease. In an attempt to better appreciate the relevance of a widely used mouse colon tumor (colon adenocarcinoma Co38) as a screening model for human colorectal tumors, we compared the main phase I and phase II drug-metabolizing enzyme systems in both tumoral and nontumoral colon tissues. The following enzymes were assayed by Western blot: cytochromes P-450 (1A1/A2, 2B1/B2, 2C, 2E1, and 3A), epoxide hydrolase, and glutathione-S-transferases (GST-alpha, -mu, and -pi). The activities of the following enzymes or cofactors were determined by spectrophotometric or fluorometric assays: total cytochrome P-450, 1-chloro-2,4-dinitrobenzene-GST, selenium-independent glutathione peroxidase, 3,4-dichloronitrobenzene-GST, ethacrynic acid-GST, total glutathione, epoxide hydrolase, UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase. Results obtained by Western blot showed that mouse colon adenocarcinoma Co38 did not express any of the probed cytochromes P-450, whereas human colorectal tumors expressed only low levels of cytochrome P-450 3A. GST-alpha and GST-pi were detected in all tumoral and nontumoral tissues of both species. The neutral GST-mu was expressed in all murine tissues investigated and was found to be polymorphic in human tissues. For human peritumoral and tumoral colorectal tissues there was no significant difference between GST isoenzyme levels, whereas mouse colon adenocarcinoma Co38 had a lower expression of GST-mu and GST-pi, compared to normal mouse colon. Enzymatic activities for glutathione peroxidase, 3,4-dichloronitrobenzene-GST, and ethacrynic acid-GST confirmed the Western blot results for GST-alpha, GST-mu, and GST-pi, respectively. Total GSH levels were similar between murine and human tumors but were 3-fold higher in human tumors than in peritumoral tissues, whereas they were 7-fold lower in mouse colon tumor Co38, compared to normal mouse colon. Epoxide hydrolase was not expressed in either mouse colon adenocarcinoma Co38 or normal mouse colon tissues, whereas it was expressed in human colon peritumoral and tumoral tissues at similar levels. No significant difference was observed between human tumors and peritumoral tissues for UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase. For murine colon tissues, the conjugation pathways (UDP-glucuronosyltransferase and sulfotransferase) were lower in colon adenocarcinoma Co38, whereas the converse was observed for the corresponding hydrolytic enzymes (beta-glucuronidase and sulfatase).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Comparison of mouse and human colon tumors with regard to phase I and phase II drug-metabolizing enzyme systems. 142 2

RIF-1 mouse tumors express high levels of beta-glucuronidase activity relative to most normal tissues. The high activity can be exploited for targeting specific drugs preferentially to tumor tissues. In this study we examined the kinetics of 8-hydroxyquinoline (8-OHQ) accumulation in tumor and in several normal tissues resulting from the in vivo deconjugation of 8-hydroxyquinolyl-glucuronide (8-OHQ-GlcA). Tumors were acidified with D-glucose and NaHCO3 prior to the administration of 8-OHQ-GlcA; subsequently the deconjugated aglycone, 8-OHQ, accumulated preferentially in tumors and reached peak levels between 30 and 60 min after the 8-OHQ-GlcA injection. Mild hyperthermia of 30 min at 43 degrees C to the tumors further increased their peak 8-OHQ levels by a factor of 2-3. Some normal tissues, mostly kidney, liver, and colon, also accumulated 8-OHQ, but the aglycone appeared early in the normal tissues (near 30 min post-injection) and was significantly reduced by 60 min when 8-OHQ remained high in the tumor. Administration of 8-OHQ-GlcA alone, without prior tumor acidification, failed to produce measurable accumulations of 8-OHQ in tumors and in normal tissues. Tissue clearance of 8-OHQ is mediated primarily by the enzymatic reconjugation of 8-OHQ via UDP-glucuronosyltransferase (UDPGT). UDPGT activity was high in liver, kidney, and bowel, but low in the RIF tumor, spleen, muscle, and brain. Hyperthermia had only a modest effects on UDPGT activity: a heat dose of 30 min at 45 degrees C reduced activity less than 60%. Thus, preferential accumulation and prolonged retention of 8-OHQ in RIF tumors may be caused by a combination of factors: a) high tumor beta-glucuronidase activity, b) selective tumor acidification during hyperglycemia, c) low tumor UDPGT activity, and d) other factors, such as tumor blood flow.
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PMID:Tumor-targeted delivery of 8-hydroxyquinoline. 190 10

Glucuronidation by liver microsomes of 3'-azido-3'-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and beta-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent Vmax of 0.98 nmol/min/mg protein and apparent Km of 13 mM. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I50 = 1.5 mM), whereas paracetamol did not exert any effect at concentrations up to 21.5 mM. Morphine also inhibited the reaction (I50 = 2.7 mM). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 A), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.
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PMID:Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3'-azido-3'-deoxythymidine: characterization of the enzyme in human and rat liver microsomes. 211 32

Significant increases in activities of epoxide hydrolase, UDP-glucuronosyltransferase, and glutathione S-transferase, and marked reductions in cytochrome P-450 mixed-function oxidase systems occur in hyperplastic nodules induced in rat liver by chemical mutagens. In contrast, activities of both oxidative (Phase I) and conjugative (Phase II) enzymes are decreased in hepatocellular carcinomas induced by peroxisome proliferators. The present work compares alterations induced by chemical mutagens or peroxisome proliferators with changes in enzyme activities that occur in primary and secondary hepatic tumors in man. The above activities, along with beta-glucuronidase and arylsulfatase, were measured in liver samples from 6 normal livers obtained at immediate autopsy, and liver specimens obtained by surgical biopsy from the following patients: 8 with hepatomas, 5 with nonmetastatic colorectal carcinomas, and 14 with metastatic colorectal carcinomas. Cytochromes P-450MP and P-450NF in addition to epoxide hydrolase were measured by immunoquantitation. Enzymes involved in conjugation reactions were either assayed fluorometrically (UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, and sulfatase) or spectrophotometrically (glutathione S-transferase) using umbelliferyl substrates or 1-chloro-2,4-dinitrobenzene. Secondary hepatic tumors showed no significant change in drug-metabolizing enzymes, in contrast to primary hepatomas, which displayed decreases in all of the measured drug metabolizing enzymes. Arylsulfatase was markedly depressed in primary hepatomas (14% of normal values). Thus, activities of drug-metabolizing enzymes in human primary tumors resemble those associated with altered hepatic foci induced by peroxisome proliferators such as ciprofibrate. The marked decreases in sulfatase that occurred in primary but not in secondary human tumors suggest that sulfation of endogenous compounds and xenobiotics may differ in patients with primary and secondary hepatic tumors.
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PMID:Hepatic drug-metabolizing enzymes in primary and secondary tumors of human liver. 302 21

A general method for the assay of UDP-glucuronosyltransferase activity towards arylcarboxylic acids (clofibric acid, 1- and 2-naphthylacetic acid) using UDP-[U-14C] glucuronic acid in liver microsomes is described. The 14C-labelled glucuronide was separated by high-performance liquid chromatography, identified by hydrolysis by beta-glucuronidase, characterized by laser desorption mass spectrometry and quantified by scintillation counting. The coefficient of variation of the enzyme activity for the inter-assay repeatability was below 4.5%. As little as 2.5 nmol of the arylcarboxylic acid glucuronides could be detected and precisely quantified. The method was applied to the determination of the apparent kinetic constants for glucuronidation of the acids. Clofibric acid was the best substrate for UDP-glucuronosyltransferase (Vmax/KM, the ratio of the maximum initial velocity and the Michaelis-Menten constant, is 12.3). The two isomers, 1- and 2-naphthylacetic acids, were transformed at a similar rate. However, they exhibited different enzymatic affinities, as the KM values were 1.0 mM and 5.6 mM for 1- and 2-naphthylacetic acid, respectively. This indicates that the spatial organization of the substrates played a critical role in this acyl glucuronoconjugation.
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PMID:Liquid chromatographic assay for the measurement of glucuronidation of arylcarboxylic acids using uridine diphospho-[U-14C] glucuronic acid. 310 34

The distribution of glucuronidation capacity along the rat intestine was investigated using mucosal cells, isolated from the small intestine, the caecum, and the colon plus rectum. The glucuronidation capacity for 1-naphthol decreases from 787 +/- 75 (duodenum) to 128 +/- 13 (colon plus rectum) pmoles/min X mg cell protein. The ratio between 1-naphthol and morphine glucuronidation was constant throughout the intestine (7.15 +/- 0.37). The distribution of maximal activity of UDP-glucuronosyltransferase in intestinal cell homogenates follows the same pattern. The maximal activity of UDPglucose dehydrogenase in homogenates corresponds closely to the glucuronidation rate in mucosal cells. The activity of beta-glucuronidase in intestinal cell homogenates is constant along the duodenum and jejunum but increases throughout the terminal ileum, caecum, colon and rectum. Subcellular fractionation studies using marker enzymes indicate that UDPglucose dehydrogenase and beta-glucuronidase are cytosolic enzymes in intestinal mucosal cells. Although UDP-glucuronosyltransferase activity is found in both the mitochondrial and the microsomal fractions, no indications for a mitochondrial localization of this enzyme can be found. Activity in the mitochondrial fraction appears to be due to endoplasmic reticulum, associated with the mitochondrial fraction.
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PMID:Distribution of glucuronidation capacity (1-naphthol and morphine) along the rat intestine. 393 47

Metabolism of benzo[a]pyrene (BP) was studied in mouse hepatocytes isolated from uninduced animals of C57BL/6 Jacobs (B6) and C3Hf/HeHa (C3) inbred strains. Conjugates with sulphate, glucuronate and glutathione were the major products of BP biotransformation in the intact cells. Their formation was measured by determining the radioactivity incorporated from [3H]BP into the appropriate metabolite, after separation on silica gel t.l.c. plates. The conjugates were identified by their susceptibility to the action of specific degrading enzymes, arylsulphatase, beta-glucuronidase and gamma-glutamyltransferase. Effects of inhibitors of conjugation were also examined. D-Galactosamine and diethyl maleate caused approximately 50% inhibition of the formation of glucuronide and glutathione derivatives of BP, respectively. The effect of salicylamide was less specific, besides an 88% decrease in sulphation of BP metabolites, a 40% decrease in the formation of glutathione conjugates was observed in the presence of this inhibitor. In hepatocytes of B6 mouse, all the above three types of BP conjugates were formed in almost equimolar quantities. The total formation of BP conjugates was 42% higher in B6 hepatocytes than in those of C3 strain. The most significant difference (1.7-fold) was in the production of BP glucuronides, despite an absence of observable differences between these mouse strains in the activity of microsomal UDP-glucuronosyltransferase and in the rate of 1-naphthol conjugation in isolated hepatocytes. Simultaneously, 2.5-fold higher accumulation of unconjugated BP metabolites was observed in the hepatocyte suspension of B6 than C3 strain and a 1.4-fold higher activity of aryl hydrocarbon hydroxylase in hepatic microsomes of this strain. The unconjugated metabolites of BP were separated into four major fractions by h.p.l.c. The retention times of the metabolites corresponded to trans 9,10-diol; trans 7,8-diol; 9-hydroxy- and 3-hydroxy-BP. Despite quantitative differences between B6 and C3 strains of mice in BP metabolism, the same degree of covalent binding of BP metabolites to cellular DNA, was observed. The results indicate a relatively high capacity of hepatocytes from uninduced mice for conjugation of BP metabolites. Hepatocytes isolated from various strains of mice, should be useful in elucidating the role of numerous factors in metabolism and biologic activity of BP and related carcinogens.
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PMID:Formation of glucuronide, sulphate and glutathione conjugates of benzo[a]pyrene metabolites in hepatocytes isolated from inbred strains of mice. 631 54

A rapid and sensitive procedure is described for the assay of rat liver microsomal UDP-glucuronosyltransferase activity toward the bile acids chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid using the radioactively labeled bile acids as substrates. The unreacted bile acids were separated from the bile acid glucuronides formed as products of the enzymatic reactions by extraction with chloroform, leaving the bile acid glucuronides in the aqueous phases. The bile acid glucuronides were characterized by their mobilities in thin-layer chromatography and identified by their sensitivity to hydrolysis with beta-glucuronidase and inhibition of hydrolysis by the specific beta-glucuronidase inhibitor D-saccharic acid-1,4-lactone. Enzyme activities were optimal at pH 6.8 and were maximally stimulated about fourfold by the addition of the nonionic detergent Brij 58 at a concentration of 0.3 mg/mg microsomal protein. The kinetic parameters for the various bile acids as substrates were determined.
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PMID:A rapid and sensitive method for the assay of UDP-glucuronosyltransferase activity toward bile acids. 641 8

(1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl. (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline. The differences were less marked with human liver microsomes. (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats. UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment. However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form. The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver. (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E. coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.
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PMID:N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes. 643 Feb 97


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