Isolation and purification of bilirubin UDP‐glucuronyl‐transferase from rat liver

Burchell, Brian

doi:10.1016/0014-5793(80)80777-0

Cited by 69 publications

(21 citation statements)

References 22 publications

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“…We have recently purified bilirubin UDPglucuronyltransferase to apparent homogeneity, although the enzyme activity did not appear to be optimally reconstituted using phosphatidylcholine liposomes (Burchell, 1980). Thus we have searched for a better alternative.…”

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confidence: 99%

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

Burchell¹

1982

Biochemical Journal

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1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.

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confidence: 99%

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

Burchell¹

1982

Biochemical Journal

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“…Gunn rat liver microsomes do not catalyze bilirubin glucuronidation, but have UDP glucuronyl transferase activity toward several phenolic and N-linked substrates (12,13). Recently, UDP glucuronyl transferase activity toward p-nitrophenol was separated from enzyme activity toward bilirubin (14), morphine (15), and estrone (16). These findings indicate that, for clinical and research studies of bilirubin metabolism, it is best to assay UDP glucuronyl transferase using bilirubin, rather than various phenols, as substrate.…”

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confidence: 99%

Bilirubin Mono- and diglucuronide formation by human liver In vitro : Assay by high-pressure liquid chromatography

Chowdhury

et al. 1981

Hepatology

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Bilirubin diglucuronide, the major pigment in human bile is formed in two steps. Bilirubin is converted to bilirubin monoglucuronide by transfer of the glucuronosyl moiety of uridine diphosphoglucuronic acid catalyzed by the microsomal enzyme, uridine diphosphoglucuronate glucuraonosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17). Bilirubin monoglucuaronide is converted to bilirubin diglucuronide in vitro by two enzymatic mechanisms: (a) UDP glucuronyl transferase-mediated transfer of a second mole of glucuronic acid form UDP-glucuronic acid to bilirubin monoglucuronide; (b) dismutation of 2 moles of bilirubin monoglucuronide to 1 mole of bilirubin diglucuronide and 1 mole of unconjugated bilirubin, catalyzed by bilirubin monoglucuronide dismutase (bilirubin glucuronoside glucuronosyl transferase EC 2.4.1.95). Assay methods for the three enzymatic mechanisms in human liver homogenate by high-pressure liquid chromatography analysis of underivatized bilirubin tetrapyrroles have been developed. UDP glucuronyl transferase was activated in five human liver homogenates with digitonin, Triton X-100, or UDP-N-acetylglucosamine. Greatest activation was observed with Triton X-100. The pH optimum for conversion of bilirubin to bilirubin monoglucuronide was 7.4, and UDP glucuronyl transferase activity was 625 +/- 51 nmoles per 20 min per gm liver. At high initial bilirubin concentrations (342 microM), the product of UDP glucuronyl transferase assay with bilirubin as substrate was predominantly bilirubin monoglucuronide. At lower initial bilirubin concentrations (6.5 to 34 microM), up to 15% bilirubin diglucuronide was formed. Glucuronyl transferase-mediated UDP glucuronic acid-dependent conversion of bilirubin monoglucuronide to diglucuronide was assayed using UDP-14-C-glucuronic acid. The pH optimum was 7.4, and the rate was 21 +/- 7 nmoles per gm liver per 20 min. The rate of bilirubin diglucuronide formation by enzymatic dismutation of bilirubin monoglucuronide was 470 +/- 112 nmoles per gm liver per min. The pH optimum was 6.6. The products of enzymatic dismutation were of the IX alpha configuration.

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“…These components also reacted more strongly with the UDPGT-isoform-non-specific RALl and phenol/ bilirubin-UDPGT RAKI rat UDPGT antisera, which may indicate that they are homologues of the rat 1 7/-hydroxy steroid and bilirubin UDPGTs. Interestingly, as with the corresponding purification of UDPGTs from rats [36], 1-naphthol UDPGT activity was eluted from the anion-exchange matrix earlier than the bilirubin UDPGT activity, which may be another indication of a degree of evolutionary structural conservation.…”

Section: Discussionmentioning

confidence: 98%

Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa

Clarke

George

Burchell

1992

Biochemical Journal

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The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated l-naphthol, but not bilirubin or steroids, and displayed a pl of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.

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Isolation and purification of bilirubin UDP‐glucuronyl‐transferase from rat liver

Cited by 69 publications

References 22 publications

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

Bilirubin Mono- and diglucuronide formation by human liver In vitro : Assay by high-pressure liquid chromatography

Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa

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