| Project/Area Number |
18570134
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Toyama Prefectural University |
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Principal Investigator |
IKUSHIRO Shinichi Toyama Prefectural University, Faculty of Engineering, Associate professor (50244679)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,190,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Xenobiotics / UDP-glucuronosyltransferase / protein-protein interaction / 異物代謝 / グルクロン酸 |
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| Research Abstract |
UDP-glucuronosyltransferases (UGT) in ER membranes can interact to form homo- and heterodimer and the interaction may play a role in the regulation of the glucuronidation including modulation of substrate specificity. We have previously indicated the heterodimeric formation between UGT1As and UGT2B1 in rat hepatic microsomes. We will focus on the regulation mechanism of the glucuronidation via oligomeric state of UGT in microsomal membranes. Immunopurification technique and western blot analysis using UGT isozyme specific peptide antibodies showed that rat UGT1A isozymes could interact with UGT2B1 but not other UGT2B isozymes, UGT2B2, 3, 6,and 12. UGT2B1 can interact with UGT1A1, 5, and 6 but not UGT2B isozymes. To analyze the functional role of the heterodimeric UGTs, we have constructed co-expression system of rat UGT1As and UGT2B in yeast. Co-expression of UGT isozymes was achieved by using two different expression vectors, genome integration plasmid and autonomously replicating plasmid. Western blot analysis using isozyme-specific antibodies confirmed the co-expression of UGT1As with UGT2B in yeast microsomes. Immunopurification of solubilized microsomes showed that UGT2B1 but not UGT2B3 was co-eluted with UGT1A, indicating the hetero-isozyme interaction of UGTs in yeast. To identify the region of UGT2B1 required for the interaction with UGT1A, we have constructed the co-expression system of chimeric UGT2B1 and UGT2B3 with UGT1A1. Co-immunoelution of UGT1A1 with UGT2B3/2B1 but not 2B1/2B3 showed clearly the involvement of C-terminal half of UGT2B1 for the interaction. Additional chimera UGT experiment with UGT1A1 and UGT2B1 suggested dimer formation of UGT through the C-terminal domain is functionally significant.
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