Abstract
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+-glucuronide was biosynthesized by incubating imipramine with recombinant UGT1A4 and then purified with solid-phase cartridges. A reversed phase-high pressure liquid chromatography (RP-HPLC) assay method was used to directly measure the concentration of imipramine and its metabolite, imipramine N+-glucuronide, with p-nitrophenol as the internal standard. The validated method was used to characterize the activity of recombinant UGT1A4 and carry out kinetic studies on imipramine glucuronidation in vitro.
Results:
The high concentration of imipramine inhibited glucuronide conjugation, so the formula V=Vmax·S/(Km+S+S2/Ki) was used to calculate the parameters, using MATLAB software. The values of apparent Km, Ki and Vmax for imipramine glucuronidation via UGT1A4 were 1.39±0.09 mmol/L, 6.24±0.45 mmol/L and 453.81±32.12 pmol/minper mgcell homogenate (n=3), respectively.
Conclusion:
As a specific substrate of UGT1A4, imipramine was used as a convenient method to characterize the activity of recombinant UGT1A4 by using HPLC. Furthermore, the profile of imipramine glucuronidation was evaluated by using recombinant UGT1A4 in vitro.
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Project supported by the National Natural Science Foundation of China (No C30100232 and No C30225047).
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Qian, Mr., Zeng, S. Biosynthesis of imipramine glucuronide and characterization of imipramine glucuronidation catalyzed by recombinant UGT1A4. Acta Pharmacol Sin 27, 623–628 (2006). https://doi.org/10.1111/j.1745-7254.2006.00314.x
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DOI: https://doi.org/10.1111/j.1745-7254.2006.00314.x
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