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CYTOCHROME
P450
Recombinant Enzyme/Microsome Protocol & References
CYTOCHROME P450 - 3A4 RECONSTITUTED FORM
SIMPLIFIED RECONSTITUTION ASSAY
Product#: RDI-P2305
1.0 SIMPLIFIED RECONSTITUTION ASSAY
RDI distributes a kit which greatly simplifies the steps involved in reconstituting purified cytochrome P450 isozymes with their associated proteins. The amount of reconstitution mix and the volume used for an assay will depend on several factors which include the:
-Sensitivity of the analytical assay for the metabolites and products from catalysis
-Kinetic parameters of enzyme catalysis for a particular substrate and P450 if known
- Incubation period of the assay
-Solubility of the test compound or substrate
-Preference of individual research groups
Most of the details for specific substrate assays for individual isozymes
have already been published (see Table 2), 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, then the following
conditions may serve as a useful starting point (Table 1):
Table 1: General Reaction Conditions
Reaction Volume 0.1 to 1.0 ml
Cytochrome P450 Isozyme 100 pmoles
Cytochrome P450 Reductase 200 pmoles
Cytochrome b(5) 100 pmoles
Substrate Concentration 100 - 500 uM
Incubation Time 5 and 10 min
Often a preincubation period of 2 to 3 minutes is performed before the reactions
are started by the addition of the final compound (substrate, or either NADPH
or a regenerating system if preferred). 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 protein.
The supernatant can then be used directly for analysis.
2.0 SAFETY PRECAUTIONS
Normal precautions exercised in handling laboratory reagents should be followed.
3.0 MATERIALS/EQUIPMENT SUPPLIED
5X Enzyme Mix: contains a 1:2:1 molar ratio of recombinant CYP3A4 (0.5 pmol/ul), cytochrome P450 reductase, cytochrome b(5). Liposomes and other buffer components are also included.
5X Buffer
4.0 Materials required but not supplied:
-100 mM NADPH or a regenerating system
-Stock solution of test drug (usually about 10-500 mM either in water or methanol*)
- distilled deionized water
-Acetonitrile or methanol (Stop Solution)
5.0 Procedure: CYP3A4 Reconstitution Assay
For a 500 ul reaction:
1) Make up NADPH and test drug stock solution.
2) Thaw out both 5X enzyme and buffer mixes rapidly and place on ice.
3) Working on ice, add 100 ul each of the 5X mixes to a 1.5 ml microfuge tube. Then add 285 ul of water and 5 ul of sub- strate*. Mix by gently flicking of the microfuge tube.
4) Preincubate the tube at 37'C for 3 minutes and start the reaction by the addition of 10 ul of NADPH.
5) Stop the reaction at a predefined incubation period by the addition of 100 ul of acetonitrile or methanol.
6) Either perform and extraction or centrifuge for ten minutes to pellet protein before analysis. (For extraction of testosterone and nifedipine metabolites using methylene chloride, we add 33 ul of 1 M potassium phosphate, pH 7.4).
*Note: The final methanol concentration should not go beyond 1-2% in case
it inhibits the reaction.
6.0 REFERENCES
TABLE 2: Specific Substrates for Cytochrom P450 Isozymes
| Isozyme | Substrate | Reference |
|
General Assay
Reconstitution Methods |
14,15,16
3,4,13 |
|
| CPY1A1 | Ethoxyresorufin | 1 |
| CYP1A2 | Methoxyresorufin | 1 |
| CYP3A4 |
Nifedipine, Testosterone, Midazolium
Erythromycin |
2, 3, 4, 5 |
| CYP2C9 | Dichlofenac | 6, 7 |
| CYP2C19 | Mephenytoin | 7, 8, 9 |
| CYP2D6 | Bufuralol, Debrisoquine, Dextromethorphan | 7, 10 |
| CYP2E1 | N-nitrosodimethylamine, Chloroxazone | 11, 12 |
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. Leeman,T. et al.(1992) Life Science 52,29-34
7. Gentest Corporation,6 Henshaw St., Woburn,MA
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.Pearce,R.E., et al.(1996) Arch,Biochem. 331: 145-169
16.Guengerich,F.P.(1994) Analysis and characterization of enzymes,pp. 1259-1313.In: Principles and Methods of Toxicology Edited by A. Wallace Hayes,Raven Press,Ltd.,New York,NY
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phone (800) 370-2222 or (201) 584-7093
fax (978) 371-2266
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