Substrate specificity and properties of uridine diphosphate glucuronyltransferase purified to apparent homogeneity from phenobarbital-treated rat liver.

1. The purification to homogeneity of stable highly active preparations of UDP-glucuronyltransferase from liver of phenobarbital-treated rats is briefly described. 2. A single polypeptide was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, of mol.wt.57000. 3. Antiserum raised against the pure enzyme produces a single sharp precipitin line after Ouchterlony double-diffusion analysis. 4. The pure UDP-glucuronyltransferase isolated from livers of untreated and phenobarbital-pretreated rats appears to be the same enzyme. 5. The Km (UDP-glucuronic acid) of the pure enzyme is 5.4 mM. 6. The activity of the pure enzyme towards 2-aminophenol can still be activated 2-3-fold by diethylnitrosamine. 7. UDP-glucose and UDP-galacturonic acid are not substrates for the purified enzyme. 8. The final preparation catalysed the glucuronidation of 4-nitrophenol, 1-naphthol, 2-aminophenol, morphine and 2-aminobenzoate. 9. Activities towards 4-nitrophenol, 1-naphthol and 2-aminophenol were all copurified. The proposed heterogeneity of UDP-glucuronyltransferase is discussed.     

Glucuronidation in the reindeer: dietary modification in the UDP-glucuronosyltransferase activity with 4-nitrophenol, 1-naphthol and phenolphthalein as acceptors

The UDP-glucuronosyltransferase activity towards 4-nitrophenol, 1-naphthol and phenolphthalein was measured from the hepatic microsomes of the reindeer (Rangifer tarandus tarandus) after summer, autumn and winter feeding periods. The microsomes were digested with trypsin or digitonin. The UDP-glucuronosyltransferase activity with 4-nitrophenol and 1-naphthol as aglycones was lower in reindeer on winter food than in ones on summer food after trypsin and digitonin digestion. The activity towards phenolphthalein was the same in each feeding period. The different seasonal feeding affects the structure of microsomal membranes and this is reflected as modifications of the UDP-glucuronosyltransferase towards different substrates.     

Fractionation of the naringinase complex from Aspergillus terreus by dye affinity chromatography

Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).     

Adenine and hypoxanthine transport in human erythrocytes: distinct substrate effects on carrier mobility.

Transport of adenine and hypoxanthine in human erythrocytes proceeds via two mechanisms: (1) a common carrier for both nucleobases and (2) unsaturable permeation 4-5-fold faster for adenine for hypoxanthine. The latter process was resistant to inactivation by diazotized sulfanilic acid. Carrier mediated transport of both substrates was investigated using zero-trans and equilibrium exchange protocols. Adenine displayed a much higher affinity for the carrier (Km approximately 5-8 microM) than hypoxanthine (Km approximately 90-120 microM) but maximum fluxes at 25 degrees C were generally 5-10-fold lower for adenine (Vmax approximately 0.6-1.4 pmol/microliters per s) than for hypoxanthine (Vmax approximately 9-11 pmol/microliters per s). The carrier behaved symmetrically with respect to influx and efflux for both substrates. Adenine, but not hypoxanthine reduced carrier mobility more than 10-fold. The mobility of the unloaded carrier, calculated from the kinetic data of either hypoxanthine or adenine transport, was the same thus providing further evidence that these substrates share a common transporter and that their membrane transport is adequately described by the alternating conformation model of carrier-mediated transport.     

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.     

Structural changes of liver microsomes in rat during neonatal life: influence on the glucuronidation rates of various substrates

The Vmax of the membrane bound UDP-glucuronosyltransferase (UDP-GT) towards group 1 substrates (4-nitrophenol, 2-naphthol) was particularly higher in young rats than in adults. On the contrary, activity towards group 2 substrates such as borneol or testosterone was not detectable in fetus liver. The developmental pattern of UDP-GT was related to changes in lipid composition of microsomes, namely in the content in lysophosphatidylcholine which rose at birth. The phospholipid-cholesterol molar ratio also increased 2 fold from the 16th day of fetal life to the 4th day after birth. Measurement of the steady state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as determination of the order parameter S and the rotation cone angle of the fluorescent probe strongly suggested an increase in membrane fluidity in rat liver microsomes during ontogenesis.     

High-performance liquid chromatographic method combining radiochemical and ultraviolet detection for determination of low activities of uridine 5'-diphosphate-glucuronosyltransferase

A novel reversed-phase high-performance liquid chromatographic method was developed to measure UDP-glucuronosyltransferase (UGT) activity. Radiochemical and UV detection were combined in this UDP-[(14)C]glucuronic acid-utilizing method which was especially aimed at determination of low activities typical of N-glucuronidation of various amines and heterocycles. 4-Nitrophenol and levomedetomidine were used as substrates to validate this method, and applicability was tested with commonly used model substrates of N-glucuronidation, 4-aminobiphenyl and amitriptyline, and several 4-arylalkyl-1H-imidazole compounds. Detection limits were very low, 0.5-10 pmol, corresponding to UGT activities from 0.04 to 0.8 pmol/min/mg protein depending on UV absorbance of the glucuronide conjugate. The sensitivity was 10- to 100-fold compared with earlier HPLC assays using radiochemical detection. This method enabled quantitation without a reference glucuronide, and its high sensitivity allows for characterization of N-glucuronidation kinetics of various substrates. Using this method, human liver microsomal UGT activity was determined for a series of 4-arylalkyl-1H-imidazoles. Of these compounds, levomedetomidine was glucuronidated at the highest rate, 1.69 nmol/min/mg protein, using a 500 microM substrate concentration. In comparison, activities for the commonly used UGT substrates, 4-nitrophenol, 4-aminobiphenyl, and amitriptyline were 18.80, 3.23, and 0.23 nmol/min/mg protein, respectively.     

Inhibition of UDP-glucuronosyltransferase activity by possible transition-state analogues in rat-liver microsomes

A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase 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 1-naphthol, bilirubin or testosterone. 2-(1-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.     

Functional and immunochemical comparison of hepatic UDP-glucuronosyltransferases in a piscine and a mammalian species.

1. The aglycone specificity of hepatic microsomal glucuronidation was compared under uniform conditions in a fish, Pleuronectes platessa and a mammal, Rattus norvegicus, representative of the most primitive and advanced vertebrate classes. 2. Both species exhibited comparable UDP-glucuronosyltransferase (UDPGT) activity towards planar phenolic substrates (1-naphthol, 4-nitrophenol); however, plaice activity towards bulky non-planar substrates such as (-)-morphine was either 200-fold lower, or for an arylacetic acid (RS-2-phenylpropionic acid) and an aryloxyacetic acid (clofibric acid) non-detectable. 3. Conjugation of the endogenous substrates, bilirubin and steroids were 4- to 40-fold lower in the plaice than in the rat. Whilst both species formed diglucuronides of the asymmetrical bilirubin IX alpha, they displayed a reciprocal preference for the initial esterification, conjugation of the C-8 side chain predominating in the rat and of C-12 in the fish. 4. Immunoblot analysis using two polyclonal antisera preparations raised against rat UDPGTs demonstrated the presence of multiple weakly cross-reacting polypeptides in fish microsomes indicative of multiple isoforms and conservation of common structural motifs over more than 350 million years since evolutionary divergence of the mammals.     

Preparation and quantification of 3'-phosphoadenosine 5'-phospho[35S]sulfate with high specific activity

The synthesis and quantitation of the sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAP35S), prepared from inorganic [35S]sulfate and ATP, were studied. An enzymatic transfer method based upon the quantitative transfer of [35S]sulfate from PAP35S to 2-naphthol and 4-methylumbelliferone by the action of phenolsulfotransferase activity from rat brain cytosol was also developed. The 2-naphthyl[35S]sulfate or 35S-methylumbelliferone sulfate formed was isolated by polystyrene bead chromatography. This method allows the detection of between 0.1 pmol and 1 nmol/ml of PAP35S. PAP35S of high specific activity (75 Ci/mmol) was prepared by incubating ATP and carrier-free Na2 35SO4 with a 100,000g supernatant fraction from rat spleen. The product was purified by ion-exchange chromatography. The specific activity and purity of PAP35S were estimated by examining the ratios of Km values for PAP35S of the tyrosyl protein sulfotransferase present in microsomes from rat cerebral cortex. The advantage and applications of these methods for the detection of femtomole amounts, and the synthesis of large scale quantities of PAP35S with high specific activity are discussed.     

Separation by FPLC chromatofocusing of UDP-glucosyltransferases from three developmental stages of Drosophila melanogaster

Variation of UDP-glucosyltransferase activity, during Drosophila melanogaster development, was analyzed. The endogenous metabolite xanthurenic acid and the xenobiotic compounds 1-naphthol and 2-naphthol were used as substrates. Developmentally regulated differences were observed for the three substrates, suggesting the presence of UDP-glucosyltransferase isoenzymes. This was further confirmed by FPLC chromatofocusing on a Mono P column: seven peaks of UDP-glucosyltransferase activity (pHs: ≥6.3, 5.8, 5.5, 4.9, 4.5, 4.2, ≤4.0) with either single or overlapping substrate specificity were detected. A single xanthurenic acid:UDP-glucosyltransferase activity (pl 5.8) was found throughout development. In contrast, a gradual increase in the number of 2-napthol:UDP-glucosyltransferase isoenzymes (pl from 6.3 to 4.0) was observed during development, whereas no isoenzymes specific for 1-naphthol were resolved. Based on the distribution and substrate specificity of the eluted peaks in the three developmental stages analyzed, the presence of seven or possibly eight UDP-glucosyltransferase isoenzymes is proposed. Arch. Insect Biochem. Physiol. 34:347–358, 1997. © 1997 Wiley-Liss, Inc.     

Human liver folylpolyglutamate synthetase: biochemical characterization and interactions with folates and folate antagonists

Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.     

Hydrolysis of pyridoxine-5'-beta-D-glucoside by a broad-specificity beta-glucosidase from mammalian tissues

Research was conducted to evaluate the ability of a broad-specificity beta-glucosidase in mammalian tissues to catalyze the hydrolytic release of free pyridoxine from pyridoxine-5'-beta-D-glucoside, a naturally occurring form of vitamin B6 in plant-derived foods. Activity was detected in liver and intestinal mucosa using tritiated pyridoxine glucoside as a substrate. In the rat and guinea pig, enzyme activity was greater in intestine than in liver or kidney while even greater activity was detected in human intestinal tissue. Reaction rates were, however, low in all tissues. Hydrolysis of the synthetic substrate 4-methylumbelliferyl-beta-D-glucoside was also greatest in intestinal tissue. The characteristics of the enzymatic hydrolysis of pyridoxine glucoside to pyridoxine included: (i) most activity in the soluble tissue fraction, (ii) a pH optimum of approximately 6.0, and (iii) inhibition caused by the addition of sodium taurocholate. These characteristics are very similar to those of the broad-specificity beta-glucosidase in mammalian tissues with respect to the hydrolysis of a variety of naturally occurring and synthetic substrates. The apparent Km was greater than 2 mM for pyridoxine glucoside hydrolysis by intestinal preparations of each species, which is much greater than expected intestinal concentrations derived from dietary sources. In vivo studies have indicated that the intestine is involved in the metabolic utilization of dietary pyridoxine glucoside. The results observed here suggest that an alternate process, possibly involving intestinal microorganisms, may also be involved in the in vivo hydrolysis of pyridoxine glucoside.     

Development of multiple activities of UDP-glucuronyltransferase in human liver.

UDP-glucuronyltransferase activities towards eight substrates were assayed in samples of foetal, term and adult human liver. Activities towards bilirubin, androsterone, testosterone, 1-naphthol, 4-nitrophenol and 2-aminophenol were present in foetal and term liver samples at less than 14% of adult values, whereas activity towards 5-hydroxytryptamine was present in foetal and term liver at 109 and 121% of adult values respectively. Thus a 'foetal' form of UDP-glucuronyltransferase may exist in human liver that is more restricted in substrate specificity than are those of the rat or rhesus monkey.     

Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps

The second-order rate constants (kcat/Km) for the beta-glucosidase-catalyzed hydrolysis of aryl beta-D-glucopyranosides show a bell-shaped dependence of pH. The pKas that characterize this dependence are 4.4 (delta Hion approximately equal to 0) and 6.7 (delta Hion approximately equal to 0). In D2O these pKas are increased by 0.5 (+/- 0.1) unit, but there is no solvent isotope effect on the pH-independent second-order rate constant. Nath and Rydon [Nath, R. L., & Rydon, H. N. (1954) Biochem. J. 57, 1-10] examined the kinetics of the beta-glucosidase-catalyzed hydrolysis of a series of substituted phenyl glucosides. We have extended this study to include glucosides with phenol leaving groups of pKa less than 7. Br?nsted plots for this extended series were nonlinear for both kcat/Km and kcat. Br?nsted coefficients for those compounds with leaving groups of pKa greater than 7 (for kcat/Km) or pKa greater than 8.5 (for kcat) were nearly equal to -1.0, indicating substantial negative charge buildup on the leaving group in the transition state. The nonlinearity indicates an intermediate in the reaction. This was confirmed by partitioning experiments in the presence of methanol as a competing glucose acceptor. A constant product ratio, [methyl glucoside]/[glucose], was found with aryl glucoside substrates varying over 16,000-fold in reactivity (V/K), indicative of a common intermediate. Viscosity variation (in sucrose-containing buffers) was used to probe the extent to which the beta-glucosidase reactions are diffusion-controlled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver

A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo[a]pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo[a]pyrene and 4-nitrophenol, and was designated GTM2 .     

An improved method excluding hemoglobin interferences for lysosomal hydrolase assays using colorimetric synthetic substrates, 2-(N-hexadecanoylamino)-4-nitrophenol derivatives

Artificial chromogenic substrates, derived from 2-(N-hexadecanoylamino)-4-nitrophenol, can be used to assay sphingomyelin phosphodiesterase, glucosylceramidase, or galactosylceramidase. Nevertheless, these enzymatic spectrophotometric assays cannot be realized on tissue preparations containing hemoglobin which interferes in the measurement. We present a selective extraction method of 2-(N-hexadecanoylamino)-4-nitrophenol which allows to avoid hemoglobin interference in this spectrophotometric assay of 2-(N-hexadecanoylamino)-4-nitrophenol. The solvents used have been tested to obtain on the one hand maximal absorbance and organic extraction of 2-(N-hexadecanoylamino)-4-nitrophenol, and on the other hand the minimal hemoglobin interference. None of the pure solvents studied being suitable, two solvent mixtures were selected: ethyl acetate/2-propanol (5/1) and 2-ethyl-1-hexanol/4-methyl-2-pentanone (1/1). These methods were tested to determine sphingomyelinase activity in enzymatic preparations and prove that they are available for lysosomal hydrolase assays using these colorimetric substrates.     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).     

Kinetic properties of the rat intestinal microsomal 1-naphthol:UDP-glucuronosyl transferase. Inhibition by UDP and UDP-N-acetylglucosamine

The kinetic properties of the rat intestinal microsomal 1-naphthol:UDPglucuronosyltransferase (EC 2.4.1.17) were investigated in fully activated microsomes prepared from isolated mucosal cells. The enzyme appeared to follow an ordered sequential bireactant mechanism in which 1-naphthol and UDP-glucuronic acid (UDPGlcUA) are the first and second binding substrates and UDP and 1-naphthol glucuronide the first and second products, respectively. Bisubstrate kinetic analysis yielded the following kinetic constants: Vmax = 102 +/- 6 nmol/min per mg microsomal protein, Km (UDPGlcUA) = 1.26 +/- 0.10 mM, Km (1-naphthol) = 96 +/- 10 microM and Ki (1-naphthol) = 25 +/- 7 microM. The rapid equilibrium random or ordered bireactant mechanisms, as well as the iso-Theorell-Chance mechanism, could be excluded by endproduct inhibition studies with UDP.UDP-N-acetylglucosamine (UDPGlcNAc), usually found to be an activator of UDP glucuronosyltransferase in liver microsomes, acted as a full competitive inhibitor towards UDPGlcUA in rat intestinal microsomes. With regard to 1-naphthol UDPGlcNAc exhibited a dual effect: both inhibition and activation was observed. The effect of activation by MgCl2 and Triton X-100 on the kinetic constants and the inhibition patterns of UDP and UDPGlcNAc were investigated. The results obtained suggest that latency in rat intestinal microsomes may be due to endproduct inhibition by UDP. This endproduct inhibition could be abolished by in vitro treatment with MgCl2 and Triton X-100.