UDP glucuronate

Figure 32-14. Conjugation of bilirubin with glucuronic acid. The glucuronate donor, UDP-glucuronic acid, is formed from UDP-glucose as depicted. The UDP-glucuronosyl-transferase is also called bilirubin-UGT.

The glucuronidation of bifirubin is discussed in Chapter 32 the reactions whereby xenobiotics are glu-curonidated are essentially similar. UDP-glucuronic acid is the glucuronyl donor, and a variety of glu-curonosyltransferases, present in both the endoplasmic reticulum and cytosol, are the catalysts. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobamate (a tranquilizer), phenol, and... 

For example, it is to be noted that UDP-glucuronic acid which is formed in the tissues, is used not only for polysaccharide synthesis, but also for neutralization and removal of toxic and useless materials or foreign compounds from the organism. 

HEPES (8.14 g, 34.2 mmol) and MgCl2 (0.876 g, 9.2 mmol) were dissolved in 197 mL of distilled water and the pH was adjusted to 7 with 1 m NaOH solution. To the resulting solution UDP-glucuronic acid sodium salt (4250 mg, 6.58 mmol) and a solution of... 

Glucuronidation Alcohols or phenols Carboxylic acids Amines Thiols Glucuronyltransferases (UDP-glucuronic acid) a- or 8-Glucuronides... 

This set of enzymes [EC 2.4.1.17], also known as UDP-glucuronosyltransferases, catalyzes the reaction of UDP-glucuronate with an acceptor to produce UDP and the 13-D-glucuronoside of the acceptor. This family of proteins accepts a wide range of substrates, including phenols, alcohols, amines, and fatty acids. 

Some bioactive compounds contain unusual pentoses in their molecules. This is the case of the antibiotic avilamycin, which contain a L-xylose. Quite recently Bechthold ct al. have biochemically characterized the genesis of this sugar and they have shown that enzyme AviE2 converted UDP-glucuronic acid to UDP-xylose via decarboxylation, indicating that the pentose residue of avilamycin A is derived from D-glucose and not from D-ribose [28]. 

A) Enzymatic incubation mixtures contain [U- C]UDP-glucuronic acid the derived azo pigments are separated by TLC, and the conjugated pigment is counted... 

Enzymes hydrolyzing UDP-glucuronic acid (UDP-glucuronic acid pyrophosphatase) and j8-D-glucuronosides ()8-glucuronidase) are present in microsomal material. Their activities in tissues depend on the species examined (G4, P2, W15). 

With washed microsomal preparations from the liver of guinea pig, rat, rabbit, mouse, and cat, conjugation of bilirubin also occurred at appreciable rates in the absence of added bivalent cation (P3). With digitonin-activated preparations from rat liver, glycosyl transfer rates were, respectively, 16-33%, 0-38%, and 58-78% of the values found at nearsaturation of Mg + when UDP-glucuronic acid, UDP-xylose, or UDP-glucose were assayed (F3, HIO). The great variability of the rates could point to an artifact. 

With UDP-glucuronic acid as the variable substrate and bilirubin at constant concentration, Michaelis-Menten kinetics were obeyed (A2, H2, HIO, P5, V2, W12). Rat liver microsomal preparations treated in different ways yielded apparent Km for UDP-glucuronic acid of 0.37-0.70 mM (A2, H2, HIO, V2), with albumin-bound bilirubin as the aglycon a higher value (1.66 mAf) was found in a carrier-free system (W12). These values probably do not represent Km for UDP-glucuronic acid at saturation with bilirubin (P5, V6). Under certain conditions of activation, cell extracts from livers of newborn and adult rats, wdicn tested with o-amino-... 

When appreciable cation-independent transferase activities were found (F3, HIO), double reciprocal plots of enzyme activity against concentration of added Mg + were nonlinear, but became linear when only the stimulated enzyme fractions were plotted. The apparent values so obtained were independent of the concentration of UDP-glucuronic acid, and vice versa (HIO), suggesting that Km for Mg + represents the dissociation constant of an Mg +-enzyme complex (D5). 

In determining enzyme activities, it is usually assumed that at a fixed set of so-called saturating substrate concentrations a sufficiently accurate value of F, ax is obtained. Bisubstrate kinetic analyses of UDP-glucu-ronyltransferase [assayed with bilirubin (P5) and p-nitrophenol (V6), respectively] indicate that a true measure of the amount of enzyme can be obtained only by suitable extrapolation procedures. This restriction applies in particular to bilirubin (A2, HIO, T8) and other aglycons (M15, V6) because of substrate inhibition. UDP-glucuronic acid was inhibitory at concentrations only about 10-fold higher than the apparent Km value (HIO) this was most pronounced at relatively short incubation times. Mg was noninhibitory at concentrations equal to 20 times the apparent Km values (F3, HIO). 

In the procedure of Wong (W12) bilirubin is incubated enzymatically with [U- C]UDP-glucuronic acid of known specific activity. The derived azo pigments are transferred quantitatively to a thin-layer plate and are separated. The spot of conjugated azo pigment is eluted and counted. With other radioactive UDP-sugars, extension of the procedure to the corresponding transfer processes is obvious. 

---