 |
|
 |
|
|
| Home >
Products >
Protein Tools >
Cytochrome P450 Enzymes >
hCYP3A4(reductase) |
 |
|
This product is not available for online ordering.
|
Description:
Recombinant human cytochrome P450 3A4 (hCYP3A4) has a broad array of applications ranging from the evaluation of potential pharmaceutical agents to the catalysis of synthetic reactions. Recombinant hCYP3A4 is useful for high throughput screening of drug candidates for possible toxicity, drug-drug interaction and dosage issues. Comparisons of the bacterially expressed human CYP3A4 with the related human liver enzymes give similar kinetic profiles (1). The supplied hCYP3A4 has a 10 amino acid deletion and an S18F change in the N-terminal membrane anchor region that improves expression in E.coli but has not been found to alter enzyme activity (1). Human NADPH-P450 reductase is also present to permit efficient hCYP3A4 activity.
Source:
The membrane fraction from E.coli DH5α-T1r co-expressing human CYP3A4 and human NADPH-cytochrome P450 reductase.
Enzyme Properties
Heat Inactivation:
No
Specific Activity:
8.2 pmol
Concentration
P450:
2 μM
Total Protein: 1.2 mg/ml
Storage Conditions
Storage Conditions:
100 mM KPO4
0.1 mM EDTA
20% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
Usage notes:- Upon receiving, aliquot and store at -70°C to avoid multiple freeze-thaw cycles.
For use thaw in 37°C water bath and immediately place on ice. - This enzyme was produced using the Pharmazyme™ technology licensed to NEB by PPD Discovery Inc.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
hCYP3A4 Assay Conditions::
hCYP3A4 activity was evaluated by following its conversion of testosterone to 6β-hydroxytestosterone. The reaction buffer was 50 mM potassium phosphate, (pH 7.5) containing 3.3 mM glucose-6-phosphate, 0.4 units/ml glucose-6-phosphate dehydrogenase, 3.3 mM MgCl2, 1.3 mM NADP+ and 0.1 mM testosterone. The addition of hCYP3A4 to a final concentration of 40 nM initiated the assay. At specific time points the reaction was quenched by adding methanol (at 50% of the reaction volume). Formation of 6β-hydroxytestosterone was followed by HPLC using a C18 column and a 10 mM KH2P04 buffer containing 60% methanol under isocratic conditions. Products and reactants were detected by monitoring their absorbance at 254 nm. The amount of product was determined by comparison to a standard of 6β-hydroxytestosterone.
References
- Gillam, E.M.J. et al. (1993) Arch. Biochem. Biophys., 305, 123-131.
- Iwata, H. et al. (1998) Biochem. Pharmacol., 55, 1315-1325.
- Barnes, H.J. (1996) E. Johnson and M. Waterman (Eds.), Methods Enzymol., 272, pp. 3-14. San Diego: Academic Press.
- Guengerich, F.P. (1991) J. Biol. Chem., 266, 10019-10022.
- Li, A.P. (2001) Drug Discovery Today, 6, 357-366.
Companion Products
E. coli DH5α Control Membranes
hCYP1A2 (reductase)
hCYP2C19 (reductase)
hCYP2C8 (reductase)
hCYP2D6 (reductase)
Legal
Licenses/Patents/Disclaimers:
This Product is a Pharmazyme™ isozymes, produced underlicense from PPD Discovery, Inc. (U.S. Patent Nos. 5,240,831 and 5,420,027). Pharmazyme™ is a trademark of PPD, Inc. This product is sold for research use only and not for resale in any form. Commercial use of these products may require a license from PPD Discovery, Inc., Morrisville, NC.
|
|
|
 |
|
|
