In this study we used two different preparations of pulled human liver
microsomes. The preparation obtained by differential centrifugation of pulled
human liver S9 fraction (the product of BD Gentest, lot number 3212595) is
reffered to as HLM-1. The HLM sample refered here as HLM-2 is an
InVitroCYP™ M-class 50-donor mixed gender pooled HLM preparation (lot
LFJ) obtained from BioIVT corporation (Baltimore, MD).
Incorporation of CYP2E1 into HLM was
performed by incubation of undiluted suspensions of HLM (20–25 mg/ml
protein, 10–13 mM phospholipid) in 125 mM K-Phosphate buffer containing
0,25M Sucrose with purified CYP2E1 for 16 – 20 hours at 4°C at
continuous stirring. CYP2E1 was added in the amount ranging from 0.25 to 2 molar
equivalents to the endogenous cytochrome P450 present in HLM. Following the
incubation the suspension was diluted 4–8 times with 125 mM K-Phosphate
buffer, pH 7.4 containing 0.25 M sucrose and centrifuged at 53,000 rpm (150,000
g) in an Optima TLX ultracentrifuge (Beckman Coulter Inc., Brea, CA, USA) with a
TLA100.3 rotor for 90 min at 4 °C. The pellet was resuspended in the same
buffer to the protein concentration of 15–20 mg/ml. The amount of
incorporated cytochrome P450 was calculated from the difference between the heme
protein added to the incubation media and the enzyme found in the supernatant.
According to the results of this assay, our procedure resulted in incorporation
of 96–98% of the added protein into the microsomal membrane.
microsomes. The preparation obtained by differential centrifugation of pulled
human liver S9 fraction (the product of BD Gentest, lot number 3212595) is
reffered to as HLM-1. The HLM sample refered here as HLM-2 is an
InVitroCYP™ M-class 50-donor mixed gender pooled HLM preparation (lot
LFJ) obtained from BioIVT corporation (Baltimore, MD).
performed by incubation of undiluted suspensions of HLM (20–25 mg/ml
protein, 10–13 mM phospholipid) in 125 mM K-Phosphate buffer containing
0,25M Sucrose with purified CYP2E1 for 16 – 20 hours at 4°C at
continuous stirring. CYP2E1 was added in the amount ranging from 0.25 to 2 molar
equivalents to the endogenous cytochrome P450 present in HLM. Following the
incubation the suspension was diluted 4–8 times with 125 mM K-Phosphate
buffer, pH 7.4 containing 0.25 M sucrose and centrifuged at 53,000 rpm (150,000
g) in an Optima TLX ultracentrifuge (Beckman Coulter Inc., Brea, CA, USA) with a
TLA100.3 rotor for 90 min at 4 °C. The pellet was resuspended in the same
buffer to the protein concentration of 15–20 mg/ml. The amount of
incorporated cytochrome P450 was calculated from the difference between the heme
protein added to the incubation media and the enzyme found in the supernatant.
According to the results of this assay, our procedure resulted in incorporation
of 96–98% of the added protein into the microsomal membrane.
