UDP-sugars

With fresh rat liver homogenates that were not fortified with UDP-sugar, appreciable conjugate formation of bilirubin occurred (HIO, M5). The process occurred in the absence of added Mg-+ and was inhibited by digitonin (HIO). The conjugation rates at 37°C were constant for the first 3- to 5-minute period, then decreased gradually to zero (M5). 

With bilirubin UDP-glucuronyltransferase from rat liver, Mn-+ was more (HIO), and Ca + less, stimulatory than Mg + (A2, F17, HIO). The behavior was similar when either UDP-glucose or UDP-xylose was used as the glycosyl donor (F3). Enzyme activities were also stimulated by Fe and Co (F3, HIO) Pb + activated glucuronyl transfer but was inhibitory with the other UDP-sugars. The effects of Mg +, Mn +, and Co are in accordance with work of Lucier et al. (L14) on the catalysis of glucuronyl transfer to p-nitrophenol and 1-naphthol by Triton X-100-activated and untreated microsomal material from rat liver. 

Most authors use a mixture of the same composition as the test mixture for the incubation control, except for omission of UDP-sugar. As outlined above, complete suppression of endogenous synthesis is preferred by the present authors for determination of enzyme activities. 

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. 

Figure 4 (a) Regeneration systems for UDP-sugar cofactors. E, glycosyltransferase Ey pyruvate... 

Senafi SB, Clarke DJ, Burchell B. Investigation of the substrate specificity of a cloned expressed human bilirubin UDP-glucuronosyltransferase UDP-sugar specificity and involvement in steroid and xenobiotic glucuronidation. Biochem J 1994 303(pt l) 233-240. 

HA synthase from Streptococcus equisimilis was employed for milligram-scale synthesis of HA (109). In this reaction system, UDP-sugars (UDP-GlcA and UDP-GlcNAc) were effectively regenerated by the catalyses of several enzymes, synthetic HA was produced in 90% yield. In addition, mutated HA synthase from Type A Pasteurella multocida (PmHAS 1-703 aa) was recently used for the stepwise synthesis of HA, which has a monodispersed molecular mass of up to 20 sugar units (110). 

The carbohydrate portion is synthesized from UDP-sugars that add to the... 

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