rev: June 7, 2000

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CYTOCHROME P450

References 


REFERENCES FOR CYP450 REAGENTS


Anti-CYP4A11

Powell PK, Wolf I, Lasker JM: Identification of CYP4A11 as the major laurate w-hydroxylase in human liver. Arch Biochem Biophys 335:219-226, 1996.

Powell PK, Wolf I, Jin R, Lasker JM: Metabolism of arachidonic acid to 20-hydroxy-5,8,11,14-eicosatetraenoic acid by P450 enzymes in human liver: involvement of CYP4F2 and CYP4A11. J Pharmacol Exp Ther 285:1327-1336, 1998.


Anti-CYP2C8

Lasker JM, Wester MR, Aramsombatdee E, Raucy JL. Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:16-28, 1998.

Goldstein JA, Faletto MB, Romkes-Sparks M. Sullivan T, Kitareewan S. Raucy JL, Lasker JM, Ghanayem B1. Evidence that CYP2C19 is the major (S)-mephenytoin 4'-hydroxylase in humans. Biochemistry 33:1743-52, 1994.


Anti-Human CYP2C9

Lasker JM, Wester MR, Aramsombatdee E, Raucy JL. Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:16-28, 1998.

Wester MR, Lasker JM, Johnson EF, Raucy JL: CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes. Drug Metab Dispos, in press, 1999.

Goldstein JA, Faletto MB, Romkes-Sparks M,Sullivan T, Kitereewan S, Raucy JL, Lasker JM, Ghanayem B1. Evidence that CYP2C19 is the major (S)-mephenytoin 4'-hydroxylase in humans. Biochemistry 33:1743-52, 1994.

Stearns RA, Charkravarty PK, Chen R, Chiu S-H: Biotransformationof losartan to its active carboxylic acid metabolite in human liver microsomes: role of cytochrome 2C and 3A subfamily members. Drug Metab Dispos 23:207-215, 1995.

Karam W, Goldstein J, Lasker J, Ghanayem B. Human CYP2C19 is a major omeprazole 5-hydroxylase, as demostrated with recombinant cytochrome P450 enzymes. Drug Metab Dispos 24:1081-1087, 1996.

Wang R, Liu L, Cheng H. Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine. Drug Metab Dispos 24:786-791, 1996.

Kumar G. Dubberke E, Rodrigues A, Roberts E, Dennisen J. Ifentification of cytochromes P450 involved in the human liver microsomal metabolism of the thromboxane A2 inhibitor seratrodast (ABT-001). Drug Metab Dispos 25:110-115, 1997.


Anti-Human CYP3A4

Wang RW, Newton DJ, Liu NY, Shou M, Rushmore T, Lu AYH. Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms. Drug Metab Dispos 27:167-172, 1999.

Stearns RA, Charkravarty PK, Chen R, Chiu S-H: Biotransformation of losartan to its active carboxylic acid metabolite in human liver microsomes: role of cytochrome 2C and 3A subfamily members. Drug Metab Dispos 23:207-215, 1995.

Karam W, Goldstein J, Lasker J, Ghanayem B. Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes. Drug Metab Dispos 24:1081-1087, 1996.

Wang R, Liu L, Cheng H. Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine. Drug Metab Dispos 24:786-791, 1996.

Kumar G, Dubberke E, Riodrigues A, Roberts E, Dennisen J Identification of cytochromes P450 involved in the human liver microsomal metabolism of the thromoboxane A2 inhibitor seratrodast (ABT-001). Drug Metab Dispos 25:110-115, 1997.

Wang RW, Lu AYH. Inhibitory anti-peptide antibody against human CYP3A4. Drug Metab Dispos 25:762-767, 1997.


Anti-Human CYP2C19

Lasker JM, Wester MR, Aramsombatdee E, Raucy JL. Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:16-28, 1998.

Wester MR, Lasker JM, Johnson EF, Raucy JL: CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes. Drug Metab Dispos, in press, 1999.

Wang R. Liu L, Cheng H. Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine. Drug Metab Dispos 24:786-791, 1996.


Anti-Human CYP2E1

Wang R, Liu L, Cheng H. Identification of human liver cytochrome P450 is isoforms involved in the in viro metabolism of cyclobenzaprine. Drug Metab Dispos 24:786-791, 1996.


Purified Human CYP2C9

Lasker JM, Wester MR, Aramsombatdee E, Raucy JL. Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:16-28, 1998.

Wester MR, Lasker JM, Johnson EF, Raucy JL: CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes. Drug Metab Dispos, in press, 1999.

Machinist JM, Mayer MD, Shet MS, Ferrero JL, Rodrigues AD: Identification of the human liver cytochrome P450 enzymes involved in the metabolism of zileuton (ABT-077) and its N-dehydroxzylated metabolite, Abbott-66193. Drug Metabl. Dispos. 23:1163-1174,1995.

Rodrigues AD, Kukulka MJ, Roberts EL, Ouellet D, Rodgers TR: [0--methyl 14C]Naproxen 0-demethylase activity in human liver microsomes. Evidence for the involvement of cytochrome P4501A2 and P4502C9/10. Drug Metab Dispos 24:126-136, 1996.


ANTIBODY INHIBITION ASSAYS

P450 antibodies (IgGs) are extremely powerful tools for assessing whether a particular P450 enzyme catalyzes a given reaction. In almost all cases, a polyclonal antibody to a P450 enzyme, upon binding to the enzyme, causes a dramatic (>95%) inhibition of the enzyme's metabolic activity. This holds true for purified, reconstituted P450s as well as for thos enzymes presnet in crude microsomes. For example, optimal amounts of anti-human CYP2E1 IgG inhibit CZX hydroxylation not only by purified human CYP2E1 (98%) but also by human liver microsomes (85%). To achieve this extent of inhibition: a)the antibody must be of high titre; b) sufficient amounts of IgG must be added (usually expressed as mg IgG/nmol P450) to the incubation mixture; c) the reaction being tested for inhibition must be catalyzed by only the P450 to which the antibody has been produced.


The method given below describes how to set up an antibody titration curve. In this example, the capacity of anti-CYP2A6 IgG to inhibit coumarin 7-hydroxylation by human liver microsomes is being tested. The commonly used IgG-to-P450 ratios of 0,1, 2.5, 5, and 10 mg IgG/nmol P450 are being utilized. Upon finding the IgG:P450 ratio that gives maximum inhibition of coumarin metabolism,this same ratio can also be employed with other human liver specimens.


Human liver microsomes = 10 nmol P450/ml

Preimmune IgG =              10 mg IgG/ml

Anti-CYP2A6 IgG =        10 mg IgG/ml

For the coumarin 7-hydroxylation assay, the other reaction components are 100 mM Tris-HCI (pH 7.4), 100 uM coumarin, and 1mM NADPH; the final incubation volume is 500 ul.

Tube # Microsomes IgG NADPH Other

Rx Components

 1 10ul (0.1 nmol) 100ul PI - +
 2 10ul 100ul PI - +
 3 10ul 100ul PI - +
 4 10ul 100ul PI + +
 5 10ul 100ul PI + +
 6 10ul 100ul PI + +
 7 10ul 90ul PI + 10ul Anti-2A6 + +
 8 10ul 90ul PI + 10ul Anti-2A6 + +
 9 10ul 90ul PI + 10ul Anti-2A6 + +
10 10ul 75ul PI + 25ul Anti-2A6 + +
11 10ul 75ul PI + 25ul Anti-2A6 + +
12 10ul 75ul PI + 25ul Anti-2A6 + +
13 10ul 50ul PI + 50ul Anti-2A6 + +
14 10ul 50ul PI + 50ul Anti-2A6 + +
15 10ul 50ul PI + 50ul Anti-2A6 + +
16 10ul 25ul PI + 75ul Anti-2A6 + +
17 10ul 25ul PI + 75ul Anti-2A6 + +
18 10ul 25ul PI + 75ul Anti-2A6 + +
19 10ul 100ul Anti-2A6 + +
20 10ul 100ul Anti-2A6 + +
21 10ul 100ul Anti-2A6 + +

Procedure

1. Add the above-described amounts of anti-2A6 and/or preimmune IgG to 13 X 100 mm glass tubes. Then, add liver microsomes (equivalent to 0.1 nmol P450). A minimum of 21 tubes is needed to perform each point in triplicate.

2. Incubate tubes for 3 min @ 37'C with shaking, then let stand for 10 min at room temp. Finally, place tubes back onto ice.

3. Add the remaining components (except NADPH) to the tubes.

4. Start reactions with NADPH, and incubate for 10 min @ 37'C.

5. Stop reactions as usual, and analyze for reaction product ( in this case, 7-hydroxycoumanrin).

6. Plot out rates of product formation vs IgG: P450 ratio used (see attached example).



INSTRUCTIONS FOR IMMUNOBLOTTING

NOTE: Our P450 antibody preparations are supplied at high protein concentrations (> 10 mg IgG/ml). To use at least some of these antibodies for Western blotting, it is advisable to remove an aliquot equivalent to 2.0 mg and add PBS containing 50% glycerol so that the IgG concentrations are 2.0 mg/ml. For exemple, you would dilute 138 ul anti-CYP3A4 with 862 ul of PBS/Glycerol. This antibody solution can be stored @ -20'C for use on blots without worry about freeze/thaw damage. The remaining antibody to be used in immunoinhibition assays should be stored @ -80'C.

1. For full size ( 15 X 15 cm) blots, add 100 ug of rabbit anti-human 3A4 or 200 ug rabbit anti-human 2A6 to 15-20 ml of blocking solution (e.g., Blotto). This would correspond to 50 ul and 100 ul, respectively, of the 2.0 mg IgG/ml solutions prepared in PBS/ glycerol as described above.

2. After incubating overnight at room temperature in a covered tray, discard P450 antibody solutions. Extensively wash the blot with blocking solution to remove traces of unbound 1'AB, and then incubate with an anti-rabbit IgG conjugate of your choice (e.g.goat ant-rabbit IgG-alkaline phosphatase).

We use a goat anti-rabbit IgG-Biotin X conjugate, followed by a complex of strepavidin plus HRP-Biotin X, and subsequent staining with 4-chloro-1-naphthol/hydrogen peroxide. Our immunochemical development system allows good visualization of CYP3A4 and CYP2A6 with loads of 5-10 ug microsomal protein applied to the original polyacrylamide gel. However, an anti-rabbit IgG-alkaline phosphatase conjugate of high specific activity with BCIP/NBT colorimetric staining should be at least as sensitive.





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