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Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Jiang L, Liang SC, Wang C, Ge GB, Huo XK, Qi XY, Deng S, Liu KX, Ma XC - Sci Rep (2015)

Bottom Line: Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway.However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme.DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Key Laboratory of Pharmacokinetic and Drug Transport of Liaoning, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China.

ABSTRACT
Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

No MeSH data available.


The enzyme kinetics of DACB glucuronidation at C-3 or C-16 in HLMs and UGT isoforms.(A) 3-O-glucuronidation of DACB in HLMs; (B) 3-O- glucuronidation of DACB in UGT1A4; (C) 16-O-glucuronidation of DACB in HLMs and HIMs; (D) 16-O-glucuronidation of DACB in UGT1A3 and UGT1A1.
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f4: The enzyme kinetics of DACB glucuronidation at C-3 or C-16 in HLMs and UGT isoforms.(A) 3-O-glucuronidation of DACB in HLMs; (B) 3-O- glucuronidation of DACB in UGT1A4; (C) 16-O-glucuronidation of DACB in HLMs and HIMs; (D) 16-O-glucuronidation of DACB in UGT1A3 and UGT1A1.

Mentions: To further characterize the isoform-specific biocatalysis by UGT1A3 and UGT1A4, kinetic analyses were performed for HLMs, HIMs, recombinant UGT1A3 and UGT1A4, respectively (Fig. 4). With respect to M-1 formation (3-O-glucuronidation), similar Michaelis-Menten kinetics were observed in HLMs and UGT1A4, respectively, as evidenced by the Eadie-Hofstee plots (Fig. S7). Additionally, M-1 formation in pooled HLMs showed a similar Km value relative to UGT1A4 (Table 1), suggesting that UGT1A4 was primarily responsible for the 3-O-glucuronidation of DACB. For M-2 formation (16-O-glucuronidation), the substrate inhibition kinetics were observed for HLMs and UGT1A3, respectively (Figs. 4C–D and S7). Although the isoform screening experiment suggested that UGT1A1 was also partly involved in M-2 formation at a high substrate concentration (600 μM), it exhibited very low glucuronosyltransferases activity and enzyme affinity. The affinity and clearance (Vmax/Km) of UGT1A1 in 16-O-glucuronidation (M-2) were less than 30 and 825 fold greater, respectively, compared with UGT1A3. These findings further showed that DACB could serve as an ideal probe to simultaneously measure the O-glucuronidation activities of UGT1A3 and UGT1A4 in complex enzyme systems.


Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Jiang L, Liang SC, Wang C, Ge GB, Huo XK, Qi XY, Deng S, Liu KX, Ma XC - Sci Rep (2015)

The enzyme kinetics of DACB glucuronidation at C-3 or C-16 in HLMs and UGT isoforms.(A) 3-O-glucuronidation of DACB in HLMs; (B) 3-O- glucuronidation of DACB in UGT1A4; (C) 16-O-glucuronidation of DACB in HLMs and HIMs; (D) 16-O-glucuronidation of DACB in UGT1A3 and UGT1A1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401096&req=5

f4: The enzyme kinetics of DACB glucuronidation at C-3 or C-16 in HLMs and UGT isoforms.(A) 3-O-glucuronidation of DACB in HLMs; (B) 3-O- glucuronidation of DACB in UGT1A4; (C) 16-O-glucuronidation of DACB in HLMs and HIMs; (D) 16-O-glucuronidation of DACB in UGT1A3 and UGT1A1.
Mentions: To further characterize the isoform-specific biocatalysis by UGT1A3 and UGT1A4, kinetic analyses were performed for HLMs, HIMs, recombinant UGT1A3 and UGT1A4, respectively (Fig. 4). With respect to M-1 formation (3-O-glucuronidation), similar Michaelis-Menten kinetics were observed in HLMs and UGT1A4, respectively, as evidenced by the Eadie-Hofstee plots (Fig. S7). Additionally, M-1 formation in pooled HLMs showed a similar Km value relative to UGT1A4 (Table 1), suggesting that UGT1A4 was primarily responsible for the 3-O-glucuronidation of DACB. For M-2 formation (16-O-glucuronidation), the substrate inhibition kinetics were observed for HLMs and UGT1A3, respectively (Figs. 4C–D and S7). Although the isoform screening experiment suggested that UGT1A1 was also partly involved in M-2 formation at a high substrate concentration (600 μM), it exhibited very low glucuronosyltransferases activity and enzyme affinity. The affinity and clearance (Vmax/Km) of UGT1A1 in 16-O-glucuronidation (M-2) were less than 30 and 825 fold greater, respectively, compared with UGT1A3. These findings further showed that DACB could serve as an ideal probe to simultaneously measure the O-glucuronidation activities of UGT1A3 and UGT1A4 in complex enzyme systems.

Bottom Line: Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway.However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme.DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Key Laboratory of Pharmacokinetic and Drug Transport of Liaoning, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China.

ABSTRACT
Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

No MeSH data available.