Characterisation of UDP-glucuronosyltransferase activity in sea turtle Chelonia mydas.

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.

Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase.

GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site.

Galactosamine prevents ethinylestradiol-induced cholestasis.

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.

Glucuronoconjugación: consecuencias toxicológicas y clínicas / Glucuronidation: toxicological and clinical consequences

La glucuronoconjugación es un proceso de gran importancia en el metabolismo de xenobióticos y sustancias endógenas, facilitando su excreción por parte del organismo. Durante mucho tiempo ha sido aceptado que los metabolitos derivados de esta vía no poseían carácter activo o reactivo. Sin embargo, en los últimos años han surgido evidencias que ponen en duda aquella creencia, con especial referencia a los acilglucurónidos de los ácidos aril 2-propiónicos, cuya inestabilidad in vivo bajo condiciones fisiológicas ha demostrado tener implicancias inmunotoxicológicas potenciales a través de su unión irreversible a las proteínas (aductos). Esta revisión considera los aspectos que han modificado la percepción de la glucuronoconjugación como una vía sin importancia toxicológica y clínica para el organismo. Por lo tanto, la pregunta que debería ser contestada podría ser: es la glucuronoconjugación una vía de producción de sustancias tóxicas tanto como un mecanismo de detoxificación?

Glucuronoconjugación: consecuencias toxicológicas y clínicas / Glucuronidation: toxicological and clinical consequences

La glucuronoconjugación es un proceso de gran importancia en el metabolismo de xenobióticos y sustancias endógenas, facilitando su excreción por parte del organismo. Durante mucho tiempo ha sido aceptado que los metabolitos derivados de esta vía no poseían carácter activo o reactivo. Sin embargo, en los últimos años han surgido evidencias que ponen en duda aquella creencia, con especial referencia a los acilglucurónidos de los ácidos aril 2-propiónicos, cuya inestabilidad in vivo bajo condiciones fisiológicas ha demostrado tener implicancias inmunotoxicológicas potenciales a través de su unión irreversible a las proteínas (aductos). Esta revisión considera los aspectos que han modificado la percepción de la glucuronoconjugación como una vía sin importancia toxicológica y clínica para el organismo. Por lo tanto, la pregunta que debería ser contestada podría ser: es la glucuronoconjugación una vía de producción de sustancias tóxicas tanto como un mecanismo de detoxificación? (AU)

Evidence for a role for the gumB and gumC gene products in the formation of xanthan from its pentasaccharide repeating unit by Xanthomonas campestris.

The biosynthesis of the extracellular polysaccharide xanthan in Xanthomonas campestris pv. campestris is directed by a cluster of 12 genes, gumB-gumM. Several xanthan-deficient mutants of the wild-type strain 8004 have previously been described which carry Tn5 insertions in this region of the chromosome. Here it is shown that the transposon insertion in one of these mutants, strain 8397, is located 15 bp upstream of the translational start site of the gumB gene. EDTA-treated cells of strain 8397 were able to synthesize the lipid-linked pentasaccharide repeating unit of xanthan from the three nucleotide sugar donors (UDP-glucose, GDP-mannose and UDP-glucuronic acid) but were unable to polymerize the pentasaccharide into mature xanthan. A subclone of the gum gene cluster carrying gumB and gumC restored xanthan production to strain 8397 to levels approximately 28% of the wild-type. In contrast, subclones carrying gumB or gumC alone were not effective. These results are discussed with reference to previous speculations, based on computer analysis, that gumB and gumC are both involved in the translocation of xanthan across the bacterial membranes.

[Increase of conjugated bilirubin in diabetics]. / Aumento de bilirrubina conjugada en diabéticos.

Three hundred patients were studied: 150 had NIDDM and the other 150 were non diabetics. Serum glucose and total and fractionated bilirrubin were measured in samples taken after night fasting. The mean glucose in the NIDDM group was 180.4 +/- 17.9 mg/dL (+/- SD) and 82.7 +/- 4.4 mg/dL in the non diabetic (p < 0.001). The levels of total bilirrubin were similar in both groups (0.84 +/- 0.04 vs 0.81 +/- 0.02 mg/dL, p > 0.40) but there were group differences in direct bilirrubin (NIDDM 0.52 +/- 0.03 vs 0.20 +/- 0.01 mg/dL, p < 0.001) and in indirect bilirrubin (NIDDM 0.32 +/- 0.03 vs 0.61 +/- 0.02 mg/dL, p < 0.001). Thus, 62% of the circulating bilirrubin was conjugated in the diabetics and only 25% in the non diabetics. We believe that at least part of the UDP-glucose pathway is altered in NIDDM and leads to an increase in the levels of glucouronic acid and, in turn, may cause a rise in direct bilirrubin at the expense of indirect bilirrubin.

Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris.

Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain.

Analysis of the interaction uridin 5'-diphosphoglucuronic acid with intestinal bilirubin UDP-glucuronyltransferase.

1. Bilirubin UDP-glucuronyltransferase Michaelis-Menten kinetic parameters for UDP-glucuronic acid were studied using native and digitonin activated microsomes obtained from rat intestinal mucosa. 2. The intestinal enzyme showed a lower apparent Vmax compared with the hepatic enzyme in both native and activated microsomes; digitonin pretreatment enhanced Vmax 4 times in the former enzyme and 2 times in the latter. 3. The affinity of UDP-glucuronic acid for the intestinal enzyme was about 2 times lower than that for the hepatic enzyme and it was not substantially modified by detergent neither in the intestine nor in the liver. 4. The lipid analysis of intestinal and hepatic microsomes showed that the former present a higher content of cholesterol and a lower phosphatidylcholine/sphingomyelin ratio than the latter, accordingly the estimation of membrane fluidity using the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that intestinal microsomes are more "rigid" than the hepatic ones. 5. These characteristics would provoke a restrictive milieu surrounding the enzyme that modifies its kinetic properties thus limiting its participation in the metabolism of bilirubin.

Hepatic bilirubin and UDP-glucuronate levels in Bolivian squirrel monkeys exhibiting fasting hyperbilirubinemia.

1. Bolivian squirrel monkeys (BoSMs), which are animal models for Gilbert's syndrome, have 40% less hepatic bilirubin UDP-glucuronyltransferase (BR-UPPG-T) activity than Brazilian squirrel monkeys (BrSMs). 2. Although fasting results in similar decreases in hepatic UDP-glucose and UDP-glucuronate levels in both simian subspecies, increased activities (55%) of BR-UDPG-T are induced only in the fasted control BrSMs, which do not exhibit the marked fasting hyperbilirubinemia (FH). 3. Total hepatic bilirubin (BR) concentrations were 50% greater in both fed and fasted BoSMs when compared to BrSMs. 4. Hepatic unconjugated BR levels increase upon fasting only in Gilbert-like BoSMs, reaching concentrations twice that observed in BrSMs. 5. Elevated hepatic BR levels in fasted BoSMs may reflect BR overproduction or inadequate glucuronidation. 6. The increased BR-UDPG-T activity induced in BrSMs during fasting could compensate in-part for the UDPGA depletion and prevent the marked FH as observed in BoSMs.

Influence of uridine diphosphoglucuronic acid on bilirubin glucuronidation in liver microsomes from normal and spironolactone-treated rats.

The formation of bilirubin glucuronides was studied using liver microsomes from normal and spironolactone (SP)-treated rats. Increasing amounts of uridine diphosphoglucuronic acid (UDPGA) were added to the incubation mixture to investigate the influence of this nucleotide on bilirubin glucuronidation. The results demonstrated that microsomal preparations from SP-treated rats had a greater capability for bilirubin glucuronidation than normal microsomes which was mostly produced at the expense of bilirubin diglucuronide (BDG) formation. On the other hand, the increase in UDPGA concentration in the incubation mixture also produced an increase of BDG formation which was more important than that of monoglucuronide. These results support the hypothesis of different subunits in the glucuronyl-transferase moiety with different substrate specificities. In addition, the inducer effect of SP on bilirubin glucuronidation is in agreement to the results obtained in living rats which were described previously.

Influence of uridine diphosphoglucuronic acid on bilirubin glucuronidation in liver microsomes from normal and spironolactone treated rats

En el presente trabajo se estudió la formación de glucuronoconjugados de bilirrubina por microsomas hepáticos de ratas normales y tratadas con el inductor espironolactona (SP). A los efectos de analizar la influencia del ácido uridina difosfoglucurónico (UDPGA) sobre dicho proceso se incorporaron cantidades crecientes de éste a las mezclas de incubación. Los microsomas de ratas tratadas con SP mostraron mayor capacidad de formación de glucurónidos de bilirrubina que los correspondientes testigos, fundamentalmente a expensas del diglucurónido (BDG). Por otro lado, al utilizar concentraciones crecientes de UDPGA en los medios de incubación, aumentó la formación de BDG en mayor proporción que la del monoglucurónido. Doicho comportamiento apoya la hipótesis de la existencia de diferentes subunidades enzimáticas glucuronizantes, con diferentes sespecificidades de sustrato. Por su parte, SP representa un efectivo inductor de la glucuronoconjugación de la bilirrubina, corroborando lo observado previamente en el animal entero (AU)

Influence of uridine diphosphoglucuronic acid on bilirubin glucuronidation in liver microsomes from normal and spironolactone treated rats

En el presente trabajo se estudió la formación de glucuronoconjugados de bilirrubina por microsomas hepáticos de ratas normales y tratadas con el inductor espironolactona (SP). A los efectos de analizar la influencia del ácido uridina difosfoglucurónico (UDPGA) sobre dicho proceso se incorporaron cantidades crecientes de éste a las mezclas de incubación. Los microsomas de ratas tratadas con SP mostraron mayor capacidad de formación de glucurónidos de bilirrubina que los correspondientes testigos, fundamentalmente a expensas del diglucurónido (BDG). Por otro lado, al utilizar concentraciones crecientes de UDPGA en los medios de incubación, aumentó la formación de BDG en mayor proporción que la del monoglucurónido. Doicho comportamiento apoya la hipótesis de la existencia de diferentes subunidades enzimáticas glucuronizantes, con diferentes sespecificidades de sustrato. Por su parte, SP representa un efectivo inductor de la glucuronoconjugación de la bilirrubina, corroborando lo observado previamente en el animal entero