Reconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochromeb5

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<ul><li><p>ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS</p><p>Vol. 348, No. 1, December 1, pp. 107115, 1997Article No. BB970378</p><p>Reconstitution Premixes for Assays Using PurifiedRecombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochrome b5</p><p>Peter M. Shaw,*,1 Natilie A. Hosea, David V. Thompson,*Janean M. Lenius,* and F. Peter Guengerich,1</p><p>*PanVera Corporation, 545 Science Drive, Madison, Wisconsin 53711; and Department of Biochemistryand Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennesee 37232-0146</p><p>Received June 17, 1997, and in revised form August 18, 1997</p><p>zymes and the development of the P450 premixes thatremain active after being stored frozen should allowThe development of enzyme and buffer premixes forfor rapid identication of novel P450 substrates andin vitro biotransformation assays is described. Theinhibitors and the development of large-scale screen-protein premixes contain a mixture of three recombi-ing assays. q 1997 Academic Pressnant human proteins, cytochrome P450 (P450) 3A4,</p><p>Key Words: cytochrome P450; reconstitution; recom-NADPH-P450 reductase, cytochrome b5 , and lipo-binant; premixes; oxidation.somes. The buffer premix contains reagents which,</p><p>when diluted, provide for optimal metabolic activitywith selected P450 3A4 substrates. P450 3A4 premixeswere competent in the oxidation of known substrates</p><p>Cytochrome P450 (P450 and/or CYP)2 enzymes (ECincluding testosterone, midazolam, nifedipine, eryth-1.14.14.1) comprise a large family of enzymes involvedromycin, benzphetamine, and amitriptyline. Premixesin the oxidative biotransformation of a wide variety ofstored at0807C for 2 months and those that underwentorganic molecules which include endogenous as well asan additional ve freeze/thaw cycles were able to hy-</p><p>droxylate testosterone at turnover rates similar to xenobiotic compounds e.g., steroids, ecosanoids, aro-freshly prepared reconstitution mixes. In addition, matic hydrocarbons, pesticides, and drugs (1, 2). In thepremixes stored unfrozen at 47C for 2 weeks showed liver, P450 enzymes are located in the endoplasmic re-no signicant loss in the rate of testosterone 6b-hy- ticulum of hepatocytes along with NADPH-P450 reduc-droxylation by P450 3A4. Premixes prepared with and tase (EC 1.6.2.4), which donates electrons for catalysis,without reduced glutathione, a component which had and cytochrome b5 (EC 4.4.2 group), which can modu-previously been found to be important for P450 3A4 late P450 catalysis in vitro, apparantly with or withoutreactions, were equally efcient at carrying out testos- electron transfer in various situations (3).terone hydroxylation under these conditions. Kinetic Currently, P450 enzymes representing ve gene fam-parameters determined for the metabolism of testos- ilies (4) are known to be expressed to some extent interone, amitriptyline, nifedipine, and benzphetamine human adult liver: CYP1A2, CYP2A6, CYP2B6, CYP-using P450 3A4 premixes were compared with human 2C8, CYP2C9, CYP2C18. CYP2C19, CYP2D6, CYP-pooled microsomes and insect microsomes prepared 2E1, CYP3A4, CYP3A5, CYP4A9, CYP4A11, andfrom cells infected with a baculovirus containing two</p><p>CYP7. Both genetic and environmental factors play acDNA inserts coding for P450 3A4 and NADPH-P450role in the types of allelic variants and levels of individ-reductase. Each format gave different Vmax and Km val-ual enzymes observed in different human (ethnic) pop-ues indicating different catalytic efciencies. Analysisulations and individuals within these groups. It hasof P450 1A2 premixes which contained different lipid</p><p>concentrations indicated that Vmax and Km could be al-tered. The availability of human P450 recombinant en- 2 Abbreviations used: CYP and P450, cytochrome P450; HPLC,</p><p>high-pressure liquid chromatography; GSH, reduced glutathione;Hepes; 4-(2-hydroxy)-1-piperazineethanesulfonic acid; Chaps, 3-[(3-</p><p>1 To whom correspondence should be addressed. Fax: (608) 233 cholamidopropyl)dimethylammonio]-1-propanesulfonate; BCA, bi-cinchoninic acid.3007. E-mail: peters@panvera.com.</p><p>1070003-9861/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.</p><p>AID ABB 0378 / 6b44$$$301 10-28-97 08:33:47 arca</p></li><li><p>108 SHAW ET AL.</p><p>been well documented that the type and levels of these ment of novel P450 3A4 premixes that can be storedand used conveniently when they are thawed. The ad-hepatic isozymes expressed by an individual can have</p><p>a major impact on the detoxication of xenobiotics, the vantage of these premixes is that they overcome boththe perception and the technical barriers associatedactivation of carcinogens, and the metabolism of a wide</p><p>variety of pharmacologically important drugs (1, 2). with performing large numbers of P450 assays usingpuried components in a reliable fashion.Members of the CYP3A subfamily, which represent ap-</p><p>proximately 30% and sometimes as much as 60% of thetotal expressed P450 in adult human liver, are known MATERIALS AND METHODSto be involved in the metabolism of numerous com-</p><p>Protein Expression and Puricationpounds including erythromycin, nifedipine, testoster-Plasmids containing modied cDNAs encoding P450 3A4 (8) orone, cyclosporine, lidocaine, imipramine, quinidine,</p><p>human cytochrome b5 (18) were transformed into Escherichia colimidazolam, verapamil, troleandomycin, and terfe-and grown in liquid expression medium. Cells were harvested andnadine (1, 2). recombinant proteins were puried with minor modication of estab-</p><p>One approach that we have used to better under- lished procedures for P450 3A4 (8, 9) and human cytochrome b5 (18).stand and characterize the molecular interactions and Human NADPH-P450 reductase was puried from insect micro-</p><p>somes infected with a baculovirus that contained a human cDNAsubstrate specicity of CYP3A members is by purica-clone (19), as previously described (6). Rat reductase was puriedtion of the native (57) and, more recently, the bacteri-from E. coli with minor modication of a method previously outlinedally expressed recombinant isozymes (8, 9). Substrate (20).</p><p>specicity of the puried enzymes is determined byreconstitution of enzyme activity. In vitro reconstitu- Reconstitution and Biotransformation Assaystion assays require combining multiple components in-</p><p>Simplied premix system. A batch of the 51 P450 3A4 proteincluding puried P450, NADPH-P450 reductase, lipidspremix (0.5 mM P450 3A4, 1.0 mM NADPH-P450 reductase, 0.5 mMthat have been sonicated to make liposomes, detergent, cytochrome b5 , 0.5 mg Chaps/ml, 0.1 mg/ml liposomes [L-a-dilauroyl-and buffer additives. Many techniques have been devel- sn-glycero-3-phosphocholine, L-a-diloleoyl-sn-glycero-3-phosphocho-</p><p>oped that use different methods to combine the compo- line, L-a-dilauroyl-sn-glycero-3-phosphoserine (1:1:1, w/w/w per mil-liliter)], 3.0 mM GSH, and 50 mM potassium Hepes, pH 7.4) and anents to form a functional enzyme complex, which is51 buffer mix (200 mM potassium Hepes, pH 7.4, 12 mM GSH, andthen used for biotransformation assays (1014). Recon-150 mM MgCl2) were prepared and frozen at 080 and 0207C in 1.0-stitution of P450 3A4 appears to be more complex than and 1.5-ml aliquots. The 51 protein premixes were made as follows</p><p>for other P450s. For example, reasonable turnovers for and are based on optimized conditions previously published (8, 9,a variety of P450 2E1 and CP450 1A2 substrates can be 12). Liposomes were prepared as previously described, in the absence</p><p>of Ar (9). The puried proteins and liposomes were mixed togetherachieved in reconstitution systems containing a singleon ice; then Hepes, GSH, and CHAPS were added, with water usedlipid, L-a-dilauroyl-sn-glycero-3-phosphocholine (15,to adjust the components to the nal concentration as described16). However, optimal activity for P450 3A4 substrates above. P450 1A2 mixes were made as described above with 0.5 mM</p><p>such as testosterone and nifedipine, but not others such P450 1A2 and 0.25 mM NADPH-P450 reductase, and without cyto-chrome b5 .as erythromycin and benzphetamine, is affected by the</p><p>A typical 300-ml biotransformation reaction was prepared by mix-composition of the liposomes, the concentration of diva-ing (on ice) 60 ml of 51 P450 3A4 protein premix and an equal volumelent cations, the presence of GSH, the buffer type, andof 51 buffer mix. H2O and substrate were then added to bring thethe ratio of the other protein components (8, 9, 12, 13). reaction almost to the working 11 concentration. The subsequent</p><p>For many researchers, the reconstitution process is addition of 6 ml of 100 mM NADPH yielded the exact 11 concentra-tion. Some experiments, indicated in the legends, used an NADPH-considered tedious, time consuming, and prone to error.generating system composed of 0.5 mM NADP/, 2 units of glucoseThis is due, in part, to the multiple pipetting required6-phosphate dehydrogenase/ml, and 10 mM glucose 6-phosphate. Un-for each of the various stages (combining all the compo- less otherwise stated (in other reactions where the nal reaction</p><p>nents for reconstitution, preincubations for complex volume was different), the concentration of the premixes remainedformation, and the subsequent biotransformation reac- as described above.tions) and because classically all these stages needed Fresh standard mix. The individual protein and buffer compo-</p><p>nents were aliquoted from stock solutions into 1.5-ml microcentrifugeto be performed on the same day due to lack of informa-tubes to achieve approximately 2.51 solutions and incubated on icetion on stability of the protein components. Histori-for 10 min. H2O and substrate were added and the mixtures werecally, microsomal P450s were never frozen in the ab- preincubated at 377C for 3 min, followed by addition of NADPH to</p><p>sence of glycerol or other polyols because of early evi- start the biotransformation reactions. The addition of substrate anddence showing the protective action of these agents NADPH adjusted the components to the same concentrations as for</p><p>the simplied mixtures.(17). In early work, a protective effect of GSH was re-ported (17), and thiols are still used in purication andstorage of P450s. Analytical Assays</p><p>In an effort to make the whole process simpler and The assay conditions varied depending on the substrate tested.more amenable for others, we have abandoned the tra- Most of the assays were modied so that reactions could be performed</p><p>in 1.5-ml microfuge tubes.ditional methodology and describe here the develop-</p><p>AID ABB 0378 / 6b44$$$301 10-28-97 08:33:47 arca</p></li><li><p>109CYTOCHROME P450 RECONSTITUTED PREMIXES</p><p>In the testosterone 6b-hydroxylation assay, the nal concentrationof testosterone was 200 mM unless otherwise indicated. The reactionswere stopped after 10 min at 377C either by addition of 90 ml of 0.66mM 11b-hydroxytestosterone, dissolved in a 2:1 mixture of CH3CNand CH3OH, or with 0.2 vol of a mixture of 1 M Na2CO3 and 2 MNaCl (pH 10.5). HPLC analysis of the products after extraction with1 ml of CH2Cl2, was performed as previously described (5).</p><p>Midazolam 1- and 4-hydroxylation assays were performed as de-scribed previously (21). Nifedipine assays were performed as de-scribed previously (5).</p><p>Benzphetamine N-demethylation was measured following the re-action in the presence of 1.0 mM d-benzphetamine. Reactions (0.5ml) were quenched by the addition of 0.2 vol of a mixture of 1 MNa2CO3 and 2 M NaCl (pH 10.5) and extracted with 2 vol of CH2Cl2.The contents of the tubes were mixed using a vortex device and thelayers were separated by centrifugation (450g for 10 min). An aliquotof the organic (lower) layer was transferred to a glass tube and driedunder N2. The residue was dissolved in 100 ml of column mobilephase and the substrate and product (N-benzylamphetamine) wereseparated on a Develosil ODS-HG-5 HPLC column using a mixture of10 mM potassium phosphate (pH 4.0):CH3CN (35:65, v/v) containing0.02% (v/v) (C2H5)3N. The ow rate was 1.5 ml per minute and detec-tion was at 254 nm.</p><p>Amitriptyline N-demethylation assays were done as described else- FIG. 1. Sodium dodecyl sulfatepolyacrylamide gel electrophoresiswhere (22). Erythromycin metabolism was determined by measuring analysis of puried proteins. Each protein (2 mg) was loaded onto aformaldehyde production, using the Nash procedure as previously 420% acrylamide preprepared gradient Novex gel (San Diego, CA)described (23, 24). Methoxyresorun O-dealkylation was measured and electrophoresed under denaturing conditions according to theas previously described (5) with slight modication. Reactions were manufacturer's instructions. Lanes 1 and 6, Mr markers (150, 100,perfomed with 2.5 pmol of P450 1A2 premix, in 120 mM sodium/ 75, 50, 35, 25, and 15 kDa); Lane 2, cytochrome b5 , Lane 3, P450potassium phosphate buffer, pH 7.7. NADPH (nal concentration 3A4; Lane 4, rat NADPH-P450 reductase, Lane 5, human NADPH-5 mM) was used to start the reaction. Fluorescence increase was P450 reductase.measured on a Beacon uoresence polarization instrument (PanVeraCorp., Madison WI) in intensity mode with excitation and emissionlters at 530 and 580 nm, respectively. Microsomal reactions wereperformed, as above, with 2 mg of protein. The purication of the components used for reconsti-</p><p>Protein concentration was estimated using the bicinchoninic acid tution experiments followed standard, well-docu-(BCA) assay (Pierce, Rockford, IL). P450 and cytochrome b5 concen-mented procedures and the purity of the proteins wastrations and NADPH-P450 reduction activity were measured as pre-</p><p>viously described (6) using extinction coefcients of 91 mM01 90% as demonstrated using sodium dodecyl sulfatecm01(De450490 for Fe2/CO vs Fe2/), 100 mM01 cm01 (De425 for Fe2/ polyacrylamide gel electrophoresis as a criterion (Fig.vs Fe3/), and 21 mM01 cm01(De550 for reduced cytochrome c), respec- 1). The specic content or activity of each of the threetively.</p><p>proteins, shown in Table I, was in good agreement withpreviously published results (8, 18, 20, 27).</p><p>ReagentsHuman pooled microsomes were purchased from XenoTech L.L.C. Stability</p><p>(Kansas City, KS). Midazolam and the 1- and 4-hydroxy metaboliteswere a kind gift from HoffmanLa Roche (Nutley, NJ). P450 3A4 Our rationale for deciding how to design the proteinBaculosomes were prepared at PanVera Corporation. Briey, Tri- premixes was dened by several criteria that had to bechoplusia ni cells were infected with a baculovirus containing cDNA met to allow exibility for P450 3A4 biotransformationinserts for human CYP3A4 and rabbit NADPH-P450 reductase as</p><p>reactions: (i) Different nal concentrations of P450previously descibed (25). The infected cells were harvested by centrif-needed to be achieved in the reaction mix, typicallyugation and microsomes prepared using a procedure described else-</p><p>where (26). All other reagents were obtained from commercial between 10 and 200 pmol P450 per milliliter, to accom-sources and were analytical grade or better. modate the expected variation in turnover rates for</p><p>different substra...</p></li></ul>

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