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CYTOCHROME P450

Recombinant Enzyme/Microsome Protocol & References

Cytochrome P450 - 3A4   ,   2D6 Microsomes
Product#: RDI-P2269   &    RDI-P2283

Page: 1 of    2

Effective Date: Nov 5, 1996

1.0     Microsome Assays General

RDI distributes insect microsomes which contain an overexpressed human cytochrome P450 and rabbit reductase. The material should be treated in a similar fashion to animal microsomes. The amount of microsomes, and the volume used for an assay will depend on several factors, including the:

- sensitivity of the analytical assay

- kinetic parameters for a particular substrate and P450, if   known

- incubation period of the assay

- solubility of the test compound or substrate

- possible requirement for cytochrome b5

- preference of individual research groups

Most of the details for specific substrate assays for individual isozymes have already been published (see Table 1 below),  however, individual laboratories have modified the assays to suit their personal preferences. If one is starting with a substrate whose kinetic parameters are not known, the following conditions may serve as a useful starting point:

General Reaction Conditions:

Reaction Volume                0.5 to 1.0 ml

Microsomal Protein           100 ug/ml

Substrate Concentration    100-500 uM

Incubation Time Points      15 and 30 min.

Often a preincubation period of 2 to 3 minutes is performed before the reactions are started by the addition of the final component. The linearity of the assay and kinetic parameters should be determined by standard methods. Reactions are stopped by adding either methanol, acetonitrile, or acid to a final concentration of 20% or greater to the microsomal incubation mixes and then placing them on ice. If an internal standard for the analytical assay is to be used, it can often be included in the stop mixture (or if not, it should be added at this point). In some cases, extraction of the metabolites from the reaction mix is useful as it enhances detection by concentrating the metabolites and removing water-soluble compounds that interfere in the analysis procedure. Typically 1.5 to 5 volumes of methylene chloride (or ethyl acetate) can be used although other salt additives which affect the pH and/or enhance the extraction procedure may be necessary. Sometimes extraction can be avoided, and in these cases (after the reaction is stopped as above), the reaction mixture is centrifuged for 10 minutes at 4'C in order to pellet the microsomes. The supernatant can then be used directly for analysis.

TABLE 1: Specific Substrates for Cytochrome P450 Isozymes

Isozyme                            Substrate                        Reference

General Assay                                                         14,15,16

Reconstitution                                                          3,4,13

Methods  

 CYP1A2                       Methoxyresorufin              1

 CYP3A4                       Nifedipine, Testosterone,   2,3,4,5

                                      Midazolam, Erythromycin

 CYP2C9                       Dichlofenac                        6

 CYP2C19                     Mephenytoin                      8,9

 CYP2D6                       Bufuralol, Debrisoquine,     10

                                      Dextromethorphan

 CYP2E1                       N-nitrosodimethylamine,      11,12

                                      Chlorzoxazone

2.0 References:

1. Burke, M.D.,et al. (1985) Biochemical Pharmacol.34:3337-3345

2. Guengerich, F.P., et al.(1986) J. Biol.Chem.261: 5051-5060

3. Brian, W.R., et al. (1990) Biochemistry 29: 11280-11292

4. Gillam,E.M.J.,et al. (1995) Arch.Biochem.Biophys.317: 374-38

5. Gorski, J.C., et al.(1994) Biochemical.Pharmacol.47:1643-1653.

6. Leman, T. et al. (1992) Life Science 52, 29-34.

8. Shimada T. et al (1986) J. Biol. Chem. 261:909-921

9. Relling, M.V. et al (1990). J. Pharmacol. Exp. Ther.252:442-447

10. Kronbach, T. et al (1987) Analytical Biochemistry, 162:24-32

11. Yang, C.S. et al (1985) Cancer Res. 45:1140-1145

12. Peter, R. et al (1990) Chem. Res. Tox. 3:566-573

13. Ingelman-Sundberg M., et al, (1996) Biochemical Biophys.Res Comm. 221:318-322

14. Methods in Enzymology, Vol. 206, (M. Waterman and E.F. Johnson, eds) Academic Press Inc 1991)

15. Guengerich, F.P., Prin & Meth. of Toxicology, Chapter 35(A. Wallace Hayes, ed.), Raven Press, Ltd, 1994

16. Pearce, R.E. et al, (1996) Arch of Biochem. & Biophys,Vol. 331, No.2:145-169

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