Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital (original) (raw)
Related papers
Biochemical Pharmacology, 1979
The ubiquitous environmental pollutant benzo(a)pyrene is metabolized by cytocbrome P-450 dependent monooxygenases to various electrophilic intermediates which either spontaneously rearrange to phenols or are converted by epoxide hydratase to dihydrodiols. These primary metabolites may recycle a second time through monooxygenases whereby probably the ultimate carcinogens are formed (l-3). Conjugation with glucuronic acid or sulfate helps to eliminate primary metabolites and thus prevents recycling. A delicate balance between toxifyfng and detoxifying reactions may be decisive for the accumulation of ultimate carcinogens.
The Journal of biological chemistry, 1990
Gunn rats lack UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. 4-Nitrophenol glucuronidation is mediated by several UDPGT isoforms that are distinct from bilirubin-UDPGT, one of which is induced after 3-methylcholanthrene (3-MC) administration in normal, but not in Gunn rats. In normal rats, 3-MC-inducible UDPGT mRNA concentration increased 15-fold in the liver and 3-fold in kidney after 3-MC (140 mg/kg) administration. Concentration of this mRNA is much lower in Gunn rat liver and kidney compared to normal. However, this mRNA was normally induced after 3-MC administration. By RNA blot hybridization, the mRNA in Gunn rat liver and kidney appeared to be of normal size. Nuclear run-on studies showed that the transcription rate for 3-MC-inducible UDPGT was 3-fold higher in Gunn rat liver and kidney than in normal and increased 3- to 5-fold after 3-MC administration. Immunotransblot studies revealed an Mr = 56,000 3-MC-inducible UDPGT in liver and kidney of normal, but no...
Proceedings of the National Academy of Sciences, 1988
A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDPglucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector Agtll. The 2.4-kilobase cDNA contained an open reading frame of 1593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of-55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of :3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase. Human hepatic microsomal UDP-glucuronosyltransferase [UDPGT; UDPglucuronate l-D-glucuronosyltransferase (acceptor-unspecific); EC 2.4.1.17] has a major role in the conjugation and production of more hydrophilic glucuronides for excretion of drugs, xenobiotics, and endogenous compounds (1). Overwhelming evidence indicates that in rat liver these glucuronidation reactions are catalyzed by a family of isoenzymes. The existence of UDPGT isoenzymes that would specifically glucuronidate bilirubin, bile acids, 5hydroxytryptamines, and drugs (such as morphine, clofibrate, and chloramphenicol) has been suggested from kinetic analysis of in vitro enzyme assays of microsomes prepared from adult (2-6) and developing (7) liver. Inherited defects of
Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes
Biochemical Pharmacology, 1990
A specific high performance liquid ~hromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol giucuronidation kinetics in human liver microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent K, and V,,,,, values were 7.37 i: 0.99mM and 4.76 zz 1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resufted in approximateIy 50% activation of microsomai paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbe~liferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Aiong with the results of previous studies of the kinetics and inhibitor profile of human Liver glucuronidation reactions (Miners ef nt., Biochem ~hur~uc~~ 37: W-671, 1988 and 37: 2839-2845, 1988), these data indicate that the model giucuronidated substrates paracetamoi, morphine and 4-methylhrmbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.
European Journal of Biochemistry, 1990
A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDPglucuronosyltransferase were tested for their inhibitory effect on glucuronidation of various substrates in a rat liver microsomal fraction. In general 4-nitrophenol glucuronidation was more effectively inhibited than that of 1naphthol, bilirubin or testosterone. 2-(l-Naphthyl)ethyl-UDP and 2,2,2-(triphenyl)ethyl-UDP were the most effective inhibitors, Their inhibitory effect was competitive towards both UDP-glucuronic acid and 4-nitrophenol. These compounds were much more effective inhibitors than UDP; therefore addition of a lipophilic group enhances the inhibitory potency of UDP. The various UDP derivatives showed differences in their abilities to inhibit the glucuronidation of the four acceptor substrates, supporting the concept that the different forms of UDP-glucuronosyl transferase have different active sites. UDP-glucuronosyltransferase (UDP-GlcA transferase) is a family of membrane-bound (iso-)enzymes that play an important role in the biotransformation of exogenous and endogenous compounds. UDP-GlcA transferases are found in all mammalian tissues but they usually have the highest activity in the liver [I, 21. We are engaged in the development of selective inhibitors for the various UDP-GlcA transferase forms. The development of isoenzyme-selective inhibitors would facilitate the study of the separate enzyme forms. If such inhibitors were also active in vivo, they could be useful in obtaining a better insight into the role of glucuronidation in the toxification of a number of drugs [3]. Furthermore, isoenzyme-selective inhibitors could be used as ligands in affinity chromatography, like the widely used UDP-hexanolamine [4]. They could also be employed to 'map' the active site of UDP-GlcA transferases about which very little is known. Very few effective inhibitors for UDP-GlcA transferases have been reported [2]. Watanabe et al. [5] found some inhibition by organophosphates and Fournel et al. [6] reported that several triphenylcarboxylic acids inhibited bilirubin UDP-GlcA transferase. Recently, Fournel-Gigleux et al. [7] published a more detailed study, in which the inhibitory potency of a series of triphenylalkanoic acids was investigated on rat liver UDP-GlcA transferases. Our strategy towards the synthesis of active-site-directed inhibitors is based on the presumed transition state for the glucuronidation reaction (Fig. 1). This reaction is believed to be a random SN2 reaction in which the UDP moiety of UDP
Microsomal UDP-Glucuronyltransferase in Rat Liver: Oxidative Activation
Basic & Clinical Pharmacology & Toxicology, 2005
Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe 3π /ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe 3π /ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe 3π /ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe 3π /ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K m and V max values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe 3π /ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe 3π /ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.
J Pharm Sci, 1995
Immobilization of hepatic uridine-5'-diphosphoglucuronosyltransferase (UDPGT) is an accepted technique for the in vitro generation of glucuronides of interest. Parikh et al. first described the immobilization of rabbit hepatic UDPGT on agarose by the modified cyanogen bromide method and demonstrated the enzymatic activity by the subsequent generation of p-nitrophenol glucuronide. Other reports have demonstrated the nonspecificity of immobilized hepatic UD-PGT against a variety of s~b s t r a t e s~~~ and the simultaneous immobilization of hepatic cytochrome P-450 and UDPGT from solubilized microsomal protein preparation^.^ The method of Parikh et al.' has been modified by the immobilization and stabilization of rat hepatic UDPGT, instead of rabbit UDPGT. The applicability of this modification was demonstrated by generation of 5-ethyl-5-(p-hydroxyphenyl)barbiturate glucuronide (p-D-ghcopyranosiduronic acid, p-( 5-ethylhexahydro-2,4,6-trioxo-5-pyrimidinyl)phenyl[ 18743-41-41], the predominate metabolite of phenobarbital in huma1-1~~9~ and rats.7,* 5-ethyl-5-phenylbarbituric acid, 5-ethyl-5-(p-hydroxyphenyllbarbituric acid, uridine-5'-diphosphoglucuronic acid sodium salt (UDPGA), D-saccharic acid 1,4-lactone, and p-glucuronidase (Type H-2; 107 200 units/mL P-glucuronidase, 4500 units/mL sulfatase) were purchased from Sigma Chemical Co. (St. Louis, MO). CNBr-activated Sepharose 4B was obtained from Pharmacia LKB (Uppsala, Sweden). All other chemicals were of analytical reagent grade. Rat hepatic UDPGT was isolated according to a modification of the methods of Parikh et a1.I and Lehman et aL4 Five Sprague Dawley rats (300-400 g; Hilltop Laboratory Animals, Scottdale, PA, pretreated with phenobarbital, 80 mgkg ip x 5 days) were decapitated and the livers were removed. All subsequent steps were performed at 4 "C. The livers were minced and homogenized in 0.25 M sucrose buffer (25% w/v, Figure 3-(a) COSY ('H--'H-NMR, 499.87 MHz) and (b) HMQC (1H-13C-NMR, 499.87 MHz) spectra of the putative glucuronide conjugate of phydroxyphenobarbital.