Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase.

Iyanagi, Takashi; Haniu, Mitsuru; Sogawa, Kazuhiro; Fujii‐Kuriyama, Yoshiaki; Watanabe, Shiro; Shively, John E.; Anan, Kazuaki

doi:10.1016/s0021-9258(18)66758-4

Cited by 188 publications

(5 citation statements)

References 47 publications

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“…Also, udpgth-2 has a cysteine at position 515 which is not present in udpgth-l. The observation that the NH2 termini contain the greatest number of differences between these two clones is consistent with the location of the most variable region between other transferase isoforms (Iyanagi et al, 1986). In a study with chimeric cDNA constructs it was shown (Mackenzie, 1990) that the selection of the acceptor substrate is controlled by the amino terminus (~300 residues).…”

Section: Resultssupporting

confidence: 56%

Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives

Ritter

Chen²,

Sheen³

et al. 1992

Biochemistry

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Two human liver UDP-glucuronosyltransferase cDNA clones, HLUG25 [Jackson, M. R., et al. (1987) Biochem. J. 242, 581-588] and UDPGTh-2 [Ritter, J. K., et al. (1990) J. Biol. Chem. 266, 7900-7906] have previously been shown to encode isozymes active in the glucuronidation of hyodeoxycholic acid (HDCA) and certain estrogen derivatives (estriols and 3,4-catechol estrogens), respectively. Here we report that the UDPGTh-2-encoded isoform (udpgth-2) and the HLUG25-encoded isoform (udpgth-1) have parallel aglycon specificities. Following expression in COS-1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and HDCA, but the udpgth-2 isozyme is 100-fold more efficient than udpgth-1. udpgth-1 and udpgth-2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which confers isoform substrate specificity. The data indicate that a high level of conservation in the amino terminus is not required for the preservation of substrate selectivity. Analysis of glucuronidation activity encoded by UDPGTh-1/UDPGTh-2 chimeric cDNAs constructed at their common restriction sites, SacI (codon 297), NcoI (codon 385), and HhaI (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 56%

Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives

Ritter

Chen²,

Sheen³

et al. 1992

Biochemistry

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“…Activity toward bilirubin was minimal. The NH2-terminal amino acid sequence was similar to that of rat hepatic microsomal p-nitrophenol UDPGT (67) but different from that reported by Yokota et al (64) for the serotonin UDPGT from rat liver microsomes.…”

Section: Rat Kidney Udpgtscontrasting

confidence: 52%

Isolation and purification of UDP-glucuronosyltransferases

Tephly¹

1990

Chem. Res. Toxicol.

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UDPGTs are members of a class of enzymes located in the endoplasmic reticulum and are encoded by a multigene family. These proteins are responsible for the glucuronidation of hundreds of xenobiotics of many chemical classes and many endogenous substances such as steroid hormones, bile acids, and bilirubin. There are a number of UDPGTs which have been identified by purification and characterization studies and a significant number which have been characterized by expression of cDNAs. On the basis of the primary structures elucidated they appear to have marked similarities (5) and are highly conserved. However, key differences in their functional properties appear to depend primarily on differences in amino acid sequences at or about the NH2-terminal area of the protein (5). Many of the UDPGTs have an extraordinarily broad substrate specificity; a few, however, are relatively specific for a given class of substrate (morphine, DT-1 UDPGTs). This places a burden on investigators to clearly identify which substrate and how many UDPGTs will be involved in any analysis of rates of glucuronidation in microsomal preparations. Caution should also be advised for extrapolation of data from hepatic microsomes of experimental animals to human hepatic microsomal preparations because human liver microsomes possess UDPGTs which are qualitatively different and, in certain cases, UDPGTs are present in human liver which are not present in lower animals.

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“…Among UGT isozymes, UGT1A6 is a major isozyme catalyzing the glucuronidation of various simple phenolic compounds such as p -nitrophenol ( p- NP) and 4-methylumbelliferone (4-MU) in the liver. This isozyme is likely to be functionally orthologous across several species including human, rat, mouse, rabbit, dog, bovine, and monkey ( − ). The UGT1A6 originates from a UGT1 gene complex by differential splicing of a unique 5‘ exon (exon 1A6) to common 3‘ exons (exons 2−5) ( , ).…”

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confidence: 99%

Activation of Glucuronidation through Reduction of a Disulfide Bond in Rat UDP-glucuronosyltransferase 1A6

2002

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UDP-glucuronosyltransferase- (UGT-) dependent glucuronidation is an important detoxification process for many endogenous and exogenous compounds in mammals. Treatment of rat hepatic microsomes with the reducing reagent dithiothreitol (DTT) resulted in a significant increase in p-nitrophenol (p-NP) glucuronidation in a time- and concentration-dependent manner. The DTT-dependent activation of glucuronidation was specific for planar phenols but not for bilirubin or testosterone without membrane perturbation of the microsomes. p-NP glucuronidation in Gunn rat hepatic microsomes lacking UGT1 isozymes was not affected by DTT, indicating that UGT1A6 in the microsomes is mainly involved in the activation. The DTT-dependent activation was inhibited by 1,6-bis(maleimido)hexane (BMH) but not by N-ethylmaleimide, indicating that cross-linking between cysteine residues in UGT1A6 is responsible for the activation. Immunoblot analysis of rat hepatic microsomes on nonreducing SDS-PAGE gels revealed that most of the UGT1A6 migrated as a monomer, suggesting that DTT could affect an intramolecular disulfide bond in the UGT1A6 that may be responsible for the activation. To identify which of the ten cysteines in UGT1A6 are involved in the disulfide bond, rat UGT1A6 wild type and a set of mutants, each with a cysteine to serine substitution, were constructed and expressed in COS cells. Treatment of COS microsomes with DTT had no effect on the activity of the wild type but BMH showed significant inhibition, suggesting that UGT1A6 expressed in COS cells may be in the reduced and activated state. Replacement of either Cys 121 or Cys 125 with serine showed insensitivity to the BMH-dependent inhibition. These results demonstrate that both Cys 121 and Cys 125 are responsible for the activation of the activity through the disulfide bond in rat UGT1A6.

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Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase.

Cited by 188 publications

References 47 publications

Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives

Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives

Isolation and purification of UDP-glucuronosyltransferases

Activation of Glucuronidation through Reduction of a Disulfide Bond in Rat UDP-glucuronosyltransferase 1A6

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