Metablism of quercetin by active human uridine diphosphate glucuronosyltransferase 1A3 ezpressed in Chinese hamster lung cells
AIM: To obtain active human recombinant uridine diphosphate glucuronosyltransferase 1A3(UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse wanscription-polymerasechain reaction (RT-PCR) using total RNA from human liver as template. The correct fragmentconfirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+),and recombinant vector was transfected into CHL ceils using a calcium phosphate method.Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then theprotein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidationactivity of UGT1A3 was determined by reverse phase-high performance liquid chromatography(RP-HPLC) coupled with diode array detector (DAD). The quercetin glucuronide was confirmedby hydrolysis with β-glucuronidase. Control experiments were performed in parallel. Thetranscription of the recombinant was also determined by RT-PCR.RESULTS: The gene was confirmed by DNA sequencing, and it was an allele (UGT1A3-3) ofUGT1A3. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies wereobtained under the selection pressure of G418. The result of RT-PCR showed transcription ofrecombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3expressed heterologously in CHL cells was in an active form, and one of gulcuronidescorresponding to quercetin was also detected.
槲皮素 尿苷二磷酸葡萄糖醛酸转移酶 聚合酶链反应 高效液相色谱法
Ya-Kun Chen Xin Li Shu-Qing Chen Su Zeng
College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310031,Zhejiang Province,China
国内会议
杭州
英文
18-26
2004-10-01（万方平台首次上网日期，不代表论文的发表时间）