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Aim:To study the profile of imipramine N~+-glucuronidation using homogenatesof recombinant uridine-5′-diphosphoglucuronosyltransferase 1A4 (UGT1A4) frombaculovirus-infected sf9 cells.Methods:Recombinant UGT1A4 was obtainedfrom sf9 cells infected with recombinant baculovirus.Imipramine N~+-glucuronidewas biosynthesized by incubating imipramine with recombinant UGT1A4 andthen purified with solid-phase cartridges.A reversed phase-high pressure liquidchromatography (RP-HPLC) assay method was used to directly measure the con-centration of imipramine and its metabolite,imipramine N~+-glucuronide,withp-nitrophenol as the internal standard.The validated method was used to charac-terize the activity of recombinant UGT1A4 and carry out kinetic studies on imi-pramine glucuronidation in vitro.Results:The high concentration of imipramineinhibited glucuronide conjugation,so the formula V=V_(max)·S/(K_m+S+S~2/K_i) was usedto calculate the parameters,using MATLAB software.The values of apparentK_m,K_i,and V_(max) for imipramine glucuronidation via UGT1A4 were 1.39±0.09mmol/L,6.24±0.45 mmol/L and 453.81±32.12 pmol/min per mg cell homogenate (n=3),respectively.Conclusion:As a specific substrate of UGT1A4,imipramine wasused as a convenient method to characterize the activity of recombinant UGT1A4by using HPLC.Furthermore,the profile of imipramine glucuronidation was evalu-ated by using recombinant UGT1A4 in vitro.
Aim: To study the profile of imipramine N ~ + -glucuronidation using homogenates of recombinant uridine-5’-diphosphoglucuronosyltransferase 1A4 (UGT1A4) from baculovirus-infected sf9 cells. Methods: Recombinant UGT1A4 was obtained from sf9 cells infected with recombinant baculovirus. Imipramine N ~ + - glucuronidewas biosynthesized by incubating imipramine with recombinant UGT1A4 and purified with solid-phase cartridges. The reversed phase-high pressure liquid chromatography (RP-HPLC) assay method was used to directly measure the con-centration of imipramine and its metabolite, imipramine N ~ + - glucuronide, withp-nitrophenol as the internal standard.The validated method was used to charac-terize the activity of recombinant UGT1A4 and carry out kinetic studies on imi-pramine glucuronidation in vitro. Results: The high concentration of imipramineinhibited glucuronide conjugation, so the formula V = V max · S / K_m + S + S ~ 2 / K_i is used to calculate the parameters, using MATLAB software.The values of apparentK_m, K_i, and V_ (max) for imipramine glucuronidation via UGT1A4 were 1.39 ± 0.09 mmol / L, 6.24 ± 0.45 mmol / L and 453.81 ± 32.12 pmol / min per mg cell homogenate (n = 3), respectively.Conclusion: As a specific substrate of UGT1A4, imipramine wasused as a convenient method to characterize the activity of recombinant UGT1A4by using HPLC. Future and the profile of imipramine glucuronidation was evaluated-ated by using recombinant UGT1A4 in vitro.