The formation of bilirubin and p-nitrophenyl glucuronides by rabbit liver

1. Glucuronide formation of bilirubin and p-nitrophenol in vitro with excess of UDP-glucuronic acid by UDP-glucuronyltransferase from livers of young and adult rabbits was studied. 2. The development of UDP-glucuronyltransferase for the two substrates followed a markedly different pattern during maturation of young rabbits, p-nitrophenol-conjugation ability being much higher at birth than that for bilirubin. 3. Mg(2+) increased bilirubin conjugation, but inhibited p-nitrophenyl glucuronide formation. 4. p-Nitrophenol acted as a potent non-competitive inhibitor for bilirubin conjugation but bilirubin did not affect p-nitrophenyl glucuronidation. 5. The enzyme for bilirubin conjugation was inactivated at pH9 during treatment with snake venom, whereas in the same preparation the activity of the corresponding enzyme for p-nitrophenol was enhanced. In addition, some solubilization of the latter enzyme could be achieved by this method. 6. The possibility of the existence of more than one enzyme system for the formation of O-glucuronides is discussed.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

Extra hepatic formation of bilirubin glucuronides in the rat

After total hepatectomy in the rat, the presence of conjugated bilirubin in the plasma has been demonstrated using an extraction technique. This is particularly striking after loading the animals with unconjugated bilirubin. The identification of bilirubin glucuronide has been achieved using thin-layer chromatography of the azopigments formed (1) with ethyl anthranilate and (2) with p-iodoaniline.     

Hydrolysis of iodothyronine glucuronides by obligately anaerobic bacteria isolated from human faecal flora

Abstract 35 bacterial strains isolated from the human faecal flora were screened for hydrolysis of the glucuronides of 3,3',5-triiodothyronine and 3,3'-diiodothyronine. Two Gram-positive obligately anaerobic strains possessed glucuronidase activity. These strains probably belong to the genus Eubacterium , but ethanol was produced in high concentrations during glucose fermentation, which makes final classification difficult. Considering the number of bacteria in the intestinal flora (> 10⁸/ml) and the biliary excretion of iodothyronine conjugates, the strains must be able to hydrolyse a major part of the total daily intestinal supply of these iodothyronine metabolites. The study extends previous observations with faecal suspensions of human and rat origin [24]. The relevance of bacterial β-glucuronidase activity for a possible enterohepatic circulation of iodothyronines is discussed.     

Uridine kinase in embryonic rat liver. Modulation of enzyme activity by 5-azacytidine

Uridine kinase activity in rat liver decreases during embryonic and postnatal development. Administration of 5-azacytidine enhances liver uridine kinase activity in adult rats, but depresses it in embryos. The liver enzymes from the foetus and the adult are precipitated at different (NH(4))(2)SO(4) concentrations although they are eluted at about the same position on chromatography on a column of Sepharose 6B.     

Effect of fatty acid chain length and saturation on formation of the acylenzyme complex by the isolated aortic enzyme fraction

Formation of acylenzyme complexes between the protein fraction isolated from pig thoracic aortas and palmitic, stearic, oleic, linoleic, linolenic and arachidic acid, was studied. The reaction brings about transformations of the periodically variable sinusoidal function of the enzyme absorbancy (energy level) depending on the kind of substrate utilized. Different changes in the enzyme energy level in the elementary process are induced by the saturated and polyunsaturated fatty acids and by oleic acid as demonstrated by the intermediate course of the function for oleylenzyme. The reaction rate constants were calculated and their negative curvilinear dependence upon molecular weight of the substrates has been shown. The differences in the acyl-enzyme reaction course and preferences are discussed with respect to the arterial metabolism and different accumulation of lipids.     

Preparation of Uridine Diphosphate-N-Acetylgalactosamine from Uridine Diphosphate-N-Acetylglucosamine by Using Microbial Enzymes

A method was developed for the large scale preparation of uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by means of microbial enzymes. With Bacillus subtilis cell-free extract as a source of UDP-GlcNAc 4-epimerase, about 35% of the UDP-GlcNAc added was converted to UDP-GalNAc. After the residual UDP-GlcNAc was degraded to uridine triphosphate and N-acetylglucosamine-1-phosphate with a protamine-treated extract of bakers' yeast as a source of UDP-GlcNAc pyrophosphorylase, UDP-GalNAc was separated by anion-exchange column chromatography. The nucleotide was recovered by adsorption on charcoal and elution with ammoniacal ethanol. The final yield was about 100 μmol.     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.