Analysis of cytochrome P450 form specific metabolism requires use of the appropriate control preparation in order to eliminate the possibility of metabolism by enzymes native to the cell lines. Corning Life Sciences prepares control microsomes and cytosols using procedures identical to those used to prepare other microsomal products.
For P450 enzymes, control microsomes are available from both Sf9 and High Five (Hi5 or BTI-TN-5B1-4) insect cell preparations. All insect cell control microsomes are prepared from insect cells infected with wild-type baculovirus. Control preparations are also available for UGT, MAO, NAT, and CES Corning® Supersomes™ enzymes products.
Normalization of Protein Concentration
Differences in total protein concentration can affect the free concentration of substrate and hence enzyme kinetics. The available substrate concentration may be significantly lower than initially assumed for those substrates which have a large potential to bind nonspecifically to protein (J. Pharmacol. Exp. Ther. 283:46 ; Drug Metab. Dispos. 25:1359 ). Total protein concentration can be controlled and normalized in each incubation by the addition of control microsomes.
NADPH Regenerating System
NADPH is a necessary cofactor in many xenobiotic metabolism reactions. NADPH is required for the measurement of oxidase activity catalyzed by P450s, FMOs, NADPH-P450 OR, and many other oxidase enzymes. A common source of NADPH in an oxidase enzyme assay is an NADPH regenerating system which generates NADPH in situ using an enzymatic reaction. For example, glucose-6-phosphate dehydrogenase (G6PDH) will convert NADP+ to NADPH in the presence of the substrate glucose-6-phosphate (Glc-6-PO4).
The Corning Gentest™ NADPH regenerating system consists of two reagents, Solution A (NADP+ and Glc-6-PO4) and Solution B (G6PDH). Each reagent is sold separately. Combined, these two reagents form a NADPH regenerating system that can be used for all NADPH requiring oxidase assays (cDNA-expressed enzymes and liver fractions). At least 200 to 400 enzyme assays can be performed using one vial each of Solution A and B. The total number of assays that can be performed is dependent on a researcher’s experimental design.