Microsomes from baculovirus insect
cells expressing human UGT isoforms (Supersomes) were obtained from
Corning GmbH, Germany. These included microsomes from baculovirus-infected
insect cells expressing UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15
Supersomes (total protein content 5 mg/mL) prepared from cells without
the human liver UGT cDNA insert. Microsomes were stored at −80
°C until used for experiments. Microsomal preparations were diluted
in 0.1 M TRIS pH 7.4 buffer to a final protein concentration of 1
mg/mL. Compound solutions with a final concentration of 10 μM
were prepared from 1 mM DMSO stock solution in double-distilled H2O. UDPGA (25 mM) (uridine-diphosphate-glucuronic acid), 50
mM Saccharolacton, and 50 μg/mL Alamethicin stock solutions
were prepared in double-distilled H2O. For the experiment,
10 μL of 1 mg/mL microsomal preparations, 27.5 μL of H2O, 22.5 μL of 400 mM TRIS pH 7.5/40 mM MgCl2 buffer, 10 μL of Saccharolacton stock solution, and 10 μL
of Alamethicin stock solution were mixed and preincubated for 5 min
at 4 °C. Subsequently, 10 μL of 10 μM compound solution
and 10 μL of 25 mM UDPGA solution were added to achieve a total
of 100 μL incubation volume. The reaction was started by heating
to 37 °C and stopped after 15 and 30 min, respectively, by cooling
to 4 °C and adding 50 μL of 33% ACN in H2O.
Samples were centrifuged at 4000 rpm, 4 °C, and supernatants
were transferred to 96-well plates for quantification of parent compound
depletion and glucuronide formation via HPLC-MS/MS.
cells expressing human UGT isoforms (Supersomes) were obtained from
Corning GmbH, Germany. These included microsomes from baculovirus-infected
insect cells expressing UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15
Supersomes (total protein content 5 mg/mL) prepared from cells without
the human liver UGT cDNA insert. Microsomes were stored at −80
°C until used for experiments. Microsomal preparations were diluted
in 0.1 M TRIS pH 7.4 buffer to a final protein concentration of 1
mg/mL. Compound solutions with a final concentration of 10 μM
were prepared from 1 mM DMSO stock solution in double-distilled H2O. UDPGA (25 mM) (uridine-diphosphate-glucuronic acid), 50
mM Saccharolacton, and 50 μg/mL Alamethicin stock solutions
were prepared in double-distilled H2O. For the experiment,
10 μL of 1 mg/mL microsomal preparations, 27.5 μL of H2O, 22.5 μL of 400 mM TRIS pH 7.5/40 mM MgCl2 buffer, 10 μL of Saccharolacton stock solution, and 10 μL
of Alamethicin stock solution were mixed and preincubated for 5 min
at 4 °C. Subsequently, 10 μL of 10 μM compound solution
and 10 μL of 25 mM UDPGA solution were added to achieve a total
of 100 μL incubation volume. The reaction was started by heating
to 37 °C and stopped after 15 and 30 min, respectively, by cooling
to 4 °C and adding 50 μL of 33% ACN in H2O.
Samples were centrifuged at 4000 rpm, 4 °C, and supernatants
were transferred to 96-well plates for quantification of parent compound
depletion and glucuronide formation via HPLC-MS/MS.
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