UDP-glucuronosyltransferase: phospholipid dependence and properties of the reconstituted apoenzyme

Gorski, Jeffrey P.; Kasper, Charles B.

doi:10.1021/bi00615a004

Cited by 11 publications

(3 citation statements)

References 34 publications

(40 reference statements)

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“…Phosphatidylcholine gave the greatest increase in glucuronidation activity. This is in agreement with other reports (Gorski & Kasper, 1978;Tukey et al, 1979;Tukey & Tephly, 1980;Singh et al, 1981a,b;Magdalou et al, 1982), where phosphatidylcholine was shown to be the most efficient phospholipid for reconstitution of UDP-glucuronosyltransferase activity in a variety of different purified preparations. 3a-Hydroxysteroid UDPglucuronosyltransferase was routinely more responsive to phospholipid reconstitution than was 17fl-hydroxysteroid UDP-glucuronosyltransferase.…”

Section: Substrate Specificity For 17/i-hydroxysteroid 3a-hydroxystesupporting

confidence: 93%

Substrate specificity and characterization of rat liver p-nitrophenol, 3 α-hydroxysteroid and 17 β-hydroxysteroid UDP-glucuronosyltransferases

Falany¹,

Green²,

Swain³

et al. 1986

Biochemical Journal

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Purified preparations of rat liver 17-hydroxysteroid, 3-hydroxyandrogen and p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferases were further characterized as to their substrate specificities, phospholipid-dependency and physical properties. The two steroid UDP-glucuronosyltransferases were shown to exhibit strict stereospecificity with respect to the conjugation of steroids and bile acids. These enzymes have been renamed 17 beta-hydroxysteroid and 3 alpha-hydroxysteroid UDP-glucuronosyltransferase to reflect this specificity for important endogenous substrates. An endogenous substrate has not yet been identified for the p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferase. The steroid UDP-glucuronosyltransferase activities were dependent on phospholipid for maximal catalytic activity. Complete delipidation rendered the UDP-glucuronosyltransferases inactive, and enzymic activity was not restored when phospholipid was added to the reaction mixture. After partial delipidation, phosphatidylcholine was the most efficient phospholipid for restoration of enzymic activity. Partial delipidation also altered the kinetic parameters of the 3 alpha-hydroxysteroid UDP-glucuronosyltransferase. The three purified UDP-glucuronosyltransferases are separate and distinct proteins, with different amino acid compositions and peptide maps generated by limited proteolysis with Staphylococcus aureus V8 proteinase. Some similarity was observed between the amino acid composition and limited proteolytic maps of the steroid UDP-glucuronosyltransferases, suggesting they are more closely related to each other than to the p-nitrophenol UDP-glucuronosyltransferase.

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Section: Substrate Specificity For 17/i-hydroxysteroid 3a-hydroxystesupporting

confidence: 93%

Substrate specificity and characterization of rat liver p-nitrophenol, 3 α-hydroxysteroid and 17 β-hydroxysteroid UDP-glucuronosyltransferases

Falany¹,

Green²,

Swain³

et al. 1986

Biochemical Journal

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“…Another factor to be considered is the ease with which the substrate can approach the active site. As suggested by Gorski and Kasper, the substrate binding site could either be embedded in the lipid bilayer or be at the junction where the protein enters the phospholipid layer ( , ). So the aglycon needs to partition from the aqueous phase into the lipid phase.…”

Section: Discussionmentioning

confidence: 99%

Glucuronidation of Hydroxylated Polychlorinated Biphenyls (PCBs)

Tampal

Lehmler

Espandiari

et al. 2002

Chem. Res. Toxicol.

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Polychlorinated biphenyls (PCBs) may be metabolized to hydroxylated compounds. While many of these metabolites are further converted to either the glucuronic acid or the sulfate conjugates by phase II enzymes, which facilitates their excretion, some hydroxylated PCBs persist in the body. This may reflect their inability to be conjugated. A possible role of uridine diphosphate glucuronosyl transferase (UGT) in the elimination of hydroxylated metabolites of PCBs was therefore investigated. Glucuronidation studies of PCB metabolites included ones which are eliminated with relative ease and also ones which are reported to be retained in blood. Liver microsomes, prepared from male Wistar rats treated by intraperitoneal injections of phenobarbital for 3 days (400 micromol/kg/day), were used as the source of UGT. Enzyme kinetics (V(max) and K(m)) were determined for each of the metabolites. The efficiency of glucuronidation (V(max)/K(m)) was found to vary from <3 to 116 microL/min/mg and was dependent on the structure of the metabolites. Substitution of chlorine atoms on the nonhydroxylated ring greatly lowered the V(max) of the enzyme, with substitution in the meta and para positions being least favorable for enzyme activity. Steric hindrance around the hydroxyl group by chlorines on adjacent carbon atoms did not play a major role. A weak relationship between the calculated dihedral angle (planarity), pK(a), log D, and enzyme activity was determined (r(2) < 0.5). However, a stronger relationship for the surface area and surface volume of the molecule was observed (r(2) >or= 0.5). This study explains in part why some PCB metabolites persist in the body.

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“…There exist, however, substantial difficulties in extrapolation of kinetic data in micellar solutions to an in vivo situation for a membrane-bound enzyme, particularly an enzyme such as UDPglucuronosyltransferase which exhibits catalytic properties dependent on phospholipid-protein interactions (Gorski & Kasper, 1978;Magdalou et al, 1982;Singh et al, 1982). At the very least, micellar solutions provide a membrane mimetic environment into which hydrophobic substrates may partition (Armstrong et al, 1980(Armstrong et al, , 1981a.…”

Section: Transferasementioning

confidence: 99%

Stereoselectivity and regioselectivity of uridine 5'-diphosphoglucuronosyltransferase toward vicinal dihydrodiols of polycyclic aromatic hydrocarbons

Lewis¹,

Armstrong²

1983

Biochemistry

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The ability of a purified rat liver microsomal uridine-5'-diphosphoglucuronosyltransferase to catalyze the glucuronidation of stereoisomeric trans- and cis-9, 10-dihydroxy-9, 10-dihydrophenanthrenes and 4, 5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes is examined. The enzyme shows the ability to discriminate kinetically between the antipodes of trans-9, 10-dihydroxy-9, 10-dihydrophenanthrene with turnover numbers of 0.070 and 1.4 s-1 and kc/Kmapp values of 4.4 X 10(3) and 1.1 X 10(3) M-1 s-1 for the 9R, 10R and 9S, 10S stereoisomers. Glucuronidation of the nondissymmetric cis-9, 10-dihydroxy-9, 10-dihydrophenanthrene proceeds with a turnover number of 0.037 s-1 and kc/Kmapp of 18 X 10(3) M-1 s-1 to give a 60/40 mixture of the two possible diastereomeric products. Three of the four stereoisomers of 4,5-dihydroxy-4,5-dihydrobenzo[alpha] pyrene are regioselectively glucuronidated by the enzyme with a high degree of kinetic discrimination. Turnover numbers for the 4S,5S, 4R,5R, and 4S,5R stereoisomers are 4.1, 0.37, and 0.23 s-1 with kc/Kmapp values of 23.8 X 10(3), 0.23 X 10(3), and 3.15 X 10(3) M-1 s-1, respectively. The 4R,5S cis isomer is not a substrate. Enzyme-catalyzed reactions of the 4S,5S and 4S,5R isomers give exclusively (greater than or equal to 95%) the 4-glucuronide with the 4R,5R isomer giving the 5-glucuronide. The kinetic and regiochemical results indicate that the enzyme recognizes hydroxyl groups on the beta-face or bottom face of the 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes.(ABSTRACT TRUNCATED AT 250 WORDS)

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UDP-glucuronosyltransferase: phospholipid dependence and properties of the reconstituted apoenzyme

Cited by 11 publications

References 34 publications

Substrate specificity and characterization of rat liver p-nitrophenol, 3 α-hydroxysteroid and 17 β-hydroxysteroid UDP-glucuronosyltransferases

Substrate specificity and characterization of rat liver p-nitrophenol, 3 α-hydroxysteroid and 17 β-hydroxysteroid UDP-glucuronosyltransferases

Glucuronidation of Hydroxylated Polychlorinated Biphenyls (PCBs)

Stereoselectivity and regioselectivity of uridine 5'-diphosphoglucuronosyltransferase toward vicinal dihydrodiols of polycyclic aromatic hydrocarbons

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