MYEBQD? SPECMCIW F63 _ CWGCHRBME 9450 m. “FEE MECROSOMAL ' ELECTRON TRANSFORT SYSEM " A Thesis forms Degree 01.51.73. _ , mecmw STATE umsveasm ' um camsrmg ems)" ' ' ' 1975 ' THESlS amoma Bi“;- HDAG & SUNS' 800K amnm w; ‘. 4| ‘-.'m-""\DY LII . L 1-1- 4's v:—-—-———;—_ 5.- 4.... ABSTRACT ANTIBODY SPECIFICITY FOR CYTOCHROME P450 IN THE MICROSOMAL ELECTRON TRANSPORT SYSTEM BY Linda Christine Chaney The Objective of this research was twofold: 1) to present evidence for the specificity of anti-hemoprotein 3 for hemoprotein 3 and 2) to present evidence for the orien— tation of hemoprotein 3 within the microsomal membrane. The 44,000 dalton hemoprotein partially purified from microsomes isolated from phenobarbital pretreated rats was the antigen used to elicit an antibody response in rabbits. The antibody was isolated and its specificity initially examined by Dr. Welton using the technique of immunopreci- pitation. The antibody appeared to be Specific for hemo- protein 3. By using several different immunologic techniques, evidence is given to support further the specificity of anti-hemoprotein 3 for hemoprotein 3 and to determine indirectly the orientation of the hemoprotein within the microsomal membrane. The results from Ouchterlony double diffusion analyses, agglutination assays and complement Linda Christine Chaney fixation assays indicate that the antibody is specific for hemoprotein 3. The results from antibody inhibition of aminopyrine-N-demethylation imply that the active site of hemoprotein 3 is either buried within the microsomal mem- brane or protected on the exterior of the membrane by sur- rounding proteins. ANTIBODY SPECIFICITY FOR CYTOCHROME P450 IN THE MICROSOMAL ELECTRON TRANSPORT SYSTEM BY Linda Christine Chaney A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1975 C: A I. l i .3/ I \J To My Parents ii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. Steven D. Aust, my graduate advisor, for his advice, encouragement and friendship throughout the phases of my work. I would also like to thank members of my committee, Drs. John C. Speck and Pamela J. Fraker, for their assis— tance and encouragement. iii TABLE OF CONTENTS INTRODUCT I ON C O O O O 0 O O O O O O O C 0 MATERIALS AND METHODS. . . . . . . . . . . Materials . . . . . . . . . . . . . . PB and 3-MC Pretreatment of Rats. . . Preparation of Microsomal Membranes . Partial Purification P448. Immunological Techniques. . . . . . . Preparation of Antibody. . . . . of Cytochrome P450 Ouchterlony Double Diffusion Analysis. Inhibition of Aminopyrine Demethylation Activity with Anti-Hemoprotein 3 , Agglutination Assays . . . . . . Complement Fixation Assays . . . SDS Polyacrylamide Gel Electrophoresis. Other Assays. . . . . . . . . . . . . RESULTS. 0 O O O O C O O O O O O O O O 0 DISCUSSION . . . . . . . . . . . . . . . . REFERENCES APPENDIX 0 iv Page 13 13 14 15 15 15 15 22 22 23 24 34 34 37 73 88 92 Table II LIST OF TABLES Page Protocol for Complement Fixation Assays. . . 36 Summary of Results from Agglutination and Complement Fixation Studies with Respect to the Amount of HemOprotein 3 Present on 1% SDS Gels and the Specific Activity of Cyto- chrome P450. . . . . . . . . . . . . . . . . 76 Figure LIST OF FIGURES Mechanism for the Involvement of NADPH— Cytochrome c Reductase and Cytochrome P450 in the Mixed-Function Oxidase System. . Elution Profile of Immune IgG from DEAE ce11u108e. O O O 0 O O O O O O O O O O O O O Elution Profile of Fab Fragments from Carboxymethyl Cellulose. . . . . . . . . . . Titration of Guinea Pig Complement . . . . . Titration of Hemolysin . . . . . . . . . . . Titration of Anti-Hemoprotein 3. . . . . . . 1%.SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of Microsomes Isolated from Control and PB- and 3—MC-Pretreated Rats and the Cytochrome P4508 Partially Purified from Microsomes Isolated from Control and PB— and 3—MC—Pretreated Rats . . . . . . . . . . . . 1%.SDS-Polyacrylamide Gel Electrophoresis Protein Profiles of Cytochrome P450 from Rat Lung Microsomes, Pseudomonas putida, and Rabbit Liver Microsomes and Hemoprotein 3 and Anti-HemOprotein 3 . . . . . . . . . . . Ouchterlony Double Diffusion Analyses of the Precipitin Reactions Between Antibody to Hemoprotein 3 and Cytochrome P450 Isolated from Various Microsomal Sources. . . . . . . vi Page 19 21 28 30 33 40 42 44 Figure 10. 11. 12. 13. 14. 15. 16. 17. 18. LIST OF FIGURES (Continued) Ouchterlony Double Diffusion Analyses of the Precipitin Reactions Between Antibody to Hemoprotein 3 and Microsomes Isolated from Control and PB— and 3—MC—Pretreated Rats . . . . Q 0 C O 0 O O O O O O O O O O C Ouchterlony Double Diffusion Analyses of the Precipitin Reactions Between Anti- Hemoprotein 3 and Cytochrome P450 Partially Purified from Microsomes Isolated from Control and PB— and 3—MC—Pretreated Rats . . Inhibition of Aminopyrine—N—Demethylation in Microsomes Isolated from PB- and 3—MC— Pretreated Rats with Immune IgG. . . . . . . Inhibition of Aminopyrine—N—Demethylation in Microsomes Isolated from PB~Pretreated Rats with Pre-Immune and Immune Fab Frag- mentS. . . . . . . . . . . . . . . . . . . . Agglutination of Microsomes Isolated from Control and PB- and 3-MC-Pretreated Rats with Anti-HemoProtein 3. . . . . . . . . . . Agglutination of 125I—Microsomes Isolated from Control and PB— and 3—MC-Pretreated Rats and with Anti—Hemoprotein 3 . . . . . . Complement Fixation Profiles of Anti- Hemoprotein 3 with Whole Microsomes. . . . . Complement Fixation Profiles of Anti— Hemoprotein 3 with Cytochrome P450 Partially Purified from Whole Microsomes . . . . . . . Complement Fixation Profile of Rat Lung Microsomes with Anti-Hemoprotein 3 . . . . . vii Page 46 48 51 53 _56 58 61 64 66 Figure 19. 20. 21° 22. LIST OF FIGURES (Continued) Page Complement Fixation Profiles of Anti- Hemoprotein 3 with Cytochrome P450 Isolated from Rat Liver and Rabbit Liver Microsomes and.§. Eutida . . . . . . . . . . . . . . . 69 Complement Fixation Profiles of Anti— Cytochrome P450cam with Cytochrome P450 from Rat Liver and Rabbit Liver Micro- somes and g. Eutida . . . . . . . . . . . . 71 Complement Fixation Profiles of Cytochrome P450 in Whole Microsomes with Anti- Hemoprotein 3 . . . . . . . . . . . . . . . 82 Complement Fixation Profiles of Partially Purified Cytochrome P450 with Anti- Hemoprotein 3 . . . . . . . . . . . . . . . 84 viii BHT CM DEAE EDTA IgG 3-MC NADH NADP NADPH PB Reductase SDS SRBC TCA Tris LIST OF ABBREVIATIONS butylated hydroxytoluene carboxymethyl diethylaminoethyl ethylenediaminetetraacetate immunoglobulin G 3—methylcholanthrene reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate reduced nicotinamide adenine dinucleotide phosphate phendbarbital NADPH—cytochrome c reductase sodium dodecyl sulfate sheep red blood cells trichloroacetic acid tris(hydroxymethyl)aminomethane ix INTRODUCT ION The rat liver endoplasmic reticulum contains an elec— tron transport system which utilizes both NADPH and 02 in the hydroxylation of various lipophilic compounds(l—B). This microsomal mixed-function oxidase system detoxifies various lipophilic drugs, carcinogens, pesticides and xeno- biotics by making them more water soluble and therefore, more easily excreted. Two unusual properties are charac- teristic of the mixed-function oxidase system: 1) it catalyzes a wide range of reactions that can generally be classified as hydroxylation reactions(4,5) and 2) this hydroxylation activity can be induced by_ig.yigg treatment of animals with various lipophilic xendbiotics(3,6). Induction of enzymes by phendbarbital, a general inducer which increases hydroxylation activity towards most sub- strates, and 3—methylcholanthrene, a Specific inducer which stimulates activity towards a particular substrate, has been studied(3). Two microsomal proteins associated with the mixed- function oxidase electron transport chain are NADPH- cytochrome c reductase and cytochrome P450(1—3). 2 NADPH-cytochrome c reductase, first isolated and charac- terized from liver microsomes by Williams and Kamin(7), is a flavoprotein assayed by its ability to transfer electrons from NADPH to exogenous cytochrome c. Cytochrome P450, first discovered in liver microsomes by Klingenberg(8) and Garfinkel(9), is a hemoprotein. Its name is derived from a carbon monoxide difference spectrum of dithionite—reduced samples in which maximum absorbance occurs at 450 nm(10,11). The current hypothesized mechanism for involvement of both microsomal proteins in the mixed-function oxidase system is summarized in Figure 1(12). The idea that a mixed—function oxidase system could metabolize a wide array of substrates without some speci- ficity is baffling since most enzymes function with specif- ic substrates. Perhaps specificity does exist and the system does conform to the concept of substrate specificity commonly found in other systems. If, for example, animals are pretreated with PB, then induction of several enzymes in the microsomal electron transport system occurs and the system metabolizes a wide range of substrates. 0n the other hand, induction of specific enzymes by pretreatment of ani- mals with 3—MC occurs, and a specific substrate is meta— bolized. The differences Observed in drug metabolism by these inducers may be due to multiple enzyme systems .Emumwm mmmowxo coHDUGSMIUmxaE may ca omgm mEOHnooumo cam mmmuospwu o mEouzooumonmmnmz mo ucmEm>Ho>cfl on» How Emwcmnome pmuflmmzuommc ucmuuso may m3osm .ANHVmuumHHflw Eoum cmxmu .oflumamcum one Smawwm mmfiaHXO ZOHBUZDhIQfiNHS HEB ZH omflm mzommOOBMU QZ¢ mmmBUDQmm U mzommUOBMUImemz ho BZMZM>QO>ZH mma mom ZmHZ¢mUWS .H muzmflm .H musmam « 0...... +mmuom§ cum +mm / um m momum+mmsomem ~0u~+mmuomem mom _ No m . m+mm non: ~+omuom§$luul «TH-lfimuoumgmfi Tall. mmncz ”.1 5 present in the mixed—function oxidase system. Studies by various investigators have produced lines of evidence for the existence of this multiplicity. The first evidence for multiplicity was differences observed in spectral forms for cytochromes induced by pretreatment of rats with PB or 3-MC(1,2). The maximum absorbance of cytochrome P450 from microsomes in PB—pre— treated rats was 450 nm while that from microsomes in 3—MC- pretreated rats was 448 nm (cytochrome P448). Kinetic studies by Alvares,.g§.§l.(13) indicated that 3—MC-induced microsomal proteins lowered the Km for hydroxylation of 3,4-benzpyrene compared to PB—induced microsomal proteins. These experiments implied that the inducing properties of PB and 3—MC were not due entirely to the amount of enzyme stimulated but the type of enzyme synthesized. Further kinetic studies of rat liver microsomal aminopyrine demethylase activity supported the evidence of more than one system that can demethylate aminopyrine(l4,15). Recently a reconstituted mixed-function oxidase system composed of a carbon monoxide binding pigment (cytochrome P450 or P448), NADPH-dependent reductase, and lipid, was used to show that the specificity for hydroxylation resided primarily in the cytochrome fraction rather than the reduc— tase or lipid fractions(16,l7). In addition their results 6 indicated that cytochrome P450 and cytochrome P448 had different substrate specificities and were, therefore, different hemoproteins. It thus appears that specificity is imposed upon the system by the existence of multiple forms of cytochrome P450. Work by Dr. A.F. welton in our laboratory provided more evidence that multiple forms of P450 do exist(l8). By staining o.r% SDS gels with benzidine and hydrogen peroxide, three hemoproteins were identified. Pretreat- ment of rats with PB induced the hemoprotein with molecular weight 44,000 while pretreatment with 3—MC induced the hemoprotein of molecular weight 53,000. The predominant hemoprotein from control rats had a molecular weight of 50,000. The work cited above does provide evidence for multi— plicity but is not direct proof. Proof will exist only after the components have been purified to homogeneity and used in reconstituted systems to determine the speci- ficity of reactions. In the meantime, techniques to be used in the present stage of development and in the future can be develoPed. One approach is to make antibodies to the particular components and observe their interactions in the system. 7 Antibodies have been raised against NADPH—cytochrome c reductase and cytochrome P450 from various organisms. Wada, gttgl.(19) made an antibody to NADPH—cytochrome c reductase to show the participation of the microsomal electron transport in. cu—oxidation of fatty acids. An anti-reductase made by Masters(20) inhibited NADPH—cyto- chrome c reductase and ethylmorphine—Nedemethylase activ- ities in pig liver microsomes. MOre importantly, the anti— body acted as a specific inhibitor, blocking only the mixed— function oxidase system found in pig liver microsomes, not that found in bovine adrenocortical mitochondria. In 1971, experiments by Glazer(21) provided immunological evidence that substrate Specificity was determined at the level of cytochrome P450, not the reductase. An antibody to rat liver reductase inhibited NADPH-cytochrome c reductase (not NADHEcytochrome c reductase) and drug oxidation of aminopyrine—N—demethylation and aniline hydroxylation in microsomes from PB-pretreated rats and benzpyrene hydrox- ylation in microsomes from 3-MC-pretreated rats. Since the reductase was inhibited to the same degree in both systems, it was concluded that induction with PB or 3-MC altered the expression of a particular cytochrome P450 and not different types of reductase. 8 In addition to using an antibody to reductase to study the types of reactions occurring in the mixed-function oxidase system, the antibody has been used to characterize the molecular weight of the native protein(22). By immuno- precipitating reductase from detergent solubilized rat liver microsomes labelled with 1251 and analyzing the pre— cipitate by SDS gel electrophoresis and gamma counting, the molecular weight of the native reductase was estimated and compared to bromelain solubilized reductase. Antibodies to cytochrome P450 have also been prepared. Mitani, gt.§l.(23) raised antibodies against solubilized cytochrome P450 from rat liver microsomes and chick embryo livers. No cross-reactivity was Observed between anti-rat P450 and chick P450 and anti-chick P450 and rat P450. In the bacterial system Pseudomonas putida, an electron trans- port system Similar to that found in rat liver microsomes has been studied(24,25). In both systems, the terminal hydroxylase is a cytochrome P450: the terminal hydroxylase is referred to as cytochrome P450cam'because of its func— tion in the camphor methylene hydroxylase system of .P. putida. It was found that bacterial and rabbit liver microsomal P450 have Similar immmnological properties. An antibody to cytochrome P450cam cross-reacted 60%-70%'with 9 rabbit liver cytochrome P450 and inhibited benzphetamine hydroxylation in the rabbit liver microsomal system(26). Thomas(27) has made an antibody to partially purified cytochrome P448 and has Shown immunological differences between cytochrome P448 and cytochrome P450. The antibody appeared to be Specific for the hemoprotein induced by 3-MC and not PB. In all cases described above, antibodies have been made to partially purified membrane proteins and used to help elucidate structural prOpertieS and mechanisms. Although these membrane proteins have not been purified to homogeneity, they can still be used to elicit antibody reSponses. By using various immunological techniques it is possible to Show: 1) the Similarity of various antigens isolated from different sources of material; 2) the speci- ficity of the antibody: and 3) the location of antigenic Sites and their interaction with enzymatic sites. If the membrane proteins described above can be purified to homo— geneity, then the types of experiments that could be per— formed are exemplified belOW. An antibody against purified (Na+-K+)-ATPase from the outer medulla of pig kidney did not inhibit the enzyme when the antibody was exposed to the outer surface of the medulla(28). When the inner surface of the medulla was 10 exposed to the antibody, no enzymatic reaction was observed: therefore, the active site of the enzyme was present on the inner surface of the medulla. The antibody also inhibited ATPase isolated from rabbit kidney and ox brain, implying that the antigenic determinants were the same regardless of the origin of the Na+ pump. The effects on several reactions mediated by the rat brain (Na+-K+)-ATPase were studied using an antibody pre- pared against the ATPase complex(29). The antibody com— pletely inhibited the (Na+-K+)—ATPase activity but had no effect on the K+-dependentfip—nitrophenylphOSphatase activ- ity. 0n the basis of these studies, the authors have hypothesized that ATPase may be a complex composed of several components, each of which are Specific for given reactions. For example, a component for Na+-dependent formation of phosphoprotein is different from a component of a K+-dependent reaction. Thus, the different functional units have various antigenic determinants which influence partial reactions within the system. Kyte has made an antibody to native (Na+-K+)-ATPase from canine renal medulla(30,3l). His studies with the antigen-antibody complexes proved several points. 1) The antibody was specific for (Na+-K*)-ATPase: similar comple- ment fixation curves were Obtained with preparations of 11 enzymes at different stages of purification. 2) The anti- gen-antibody complexes were located on the inner surface of the plasma membrane of the intact cell. 3) The anti- bodies did not inhibit (Na+-K*)-ATPase when bound to the enzyme at saturating concentrations, implying that a dif- fusional carrier mechanism for active Na+ and K+ transport was impossible. Since various components of the mixed-function oxi— dase system have not been purified to homogeneity, it is impossible to prove the Specificity of an antibody or the effects of interaction between antibody and antigen. It is possible to present evidence that implies some struc- tural relationships and mechanisms. In our laboratory, for example, an antibody to cytochrome P450 partially purified from microsomes isolated from PB-pretreated rats has been made. It has been Shown that this cytochrome is one of three hemoproteins detected on o.r% SDS gels(l8): it cor- responds to the 44,000 dalton protein found in microsomes from PB-pretreated rats. Dr. Welton has provided evidence that the antibody immunoprecipitated only hemoprotein 3 from cytochrome P450 partially purified from liver micro- somes of PB—pretreated rats(32,33). By using this antibody in conjunction with various immunological techniques, this thesis will present evidence 12 to support further the specificity of the antibody for hemoprotein 3. Techniques used include inhibition of amino- pyrine—N-demethylation by anti-hemoprotein 3, agglutination of anti-hemoprotein 3 to various types of rat liver micro- somes and complement fixation of various cytochrome P4508 to anti-hemoprotein 3. MATERIALS AND METHODS Materials Male Sprague—Dawley rats weighing between 200-250 g and 75-100 9; were Obtained from Spartan Research Animals, Inc., Haslett, Michigan. Male New Zealand rabbits (6—8 lbs.) were obtained from the Center for Laboratory Animals Resources, Michigan State University. Sheep red blood cells were Obtained through Mr. George Good from the Department of Animal Hquandry, Michigan State University. Butylated hydroxytoluene, 3-methylcholanthrene, mer- curipapain, Trizma base, NADP—isocitrate dehydrogenaSe, isocitric acid, lactoperoxidase, bovine serum albumin, sodium dodecyl sulfate, Brilliant Blue R (Coomassie blue), nicotinamide, sodium azide, and dithiothreitol were prod- ucts of Sigma Chemical Co., St. Louis, Missouri. Glucose and phenObarbital were purchased from Merck & Company, Inc., Rahway, New Jersey. Diethylaminoethyl cellulose and carboxymethyl cellulose were Obtained from Bio—Rad Laboratories, 32nd and Griffin, Richmond, Cali- fornia. Sucrose and sodium deoxycholate were products of Schwartz—Mann Division of Becton and Co., Orangeburg, 13 14 New YOrk. Aminopyrine was Obtained through Canalco, Inc., Rockville, Maryland. Trichloroacetic acid and citric acid were products of Mallinckrodt, St. Louis, Missouri. Freund's complete adjuvant and agar (NObel Grade) were purchased from Difco Laboratories, Detroit, Michigan. Hemolysin and guinea pig complement were Obtained from Cappel Laboratories, Downington, Pennsylvania. Carrier- free Na-IZSI was Obtained from New England Nuclear, Boston, Massachusetts. Cytochrome P450cam isolated from Pseudomonas putida and the antibody againSt cytochrome P450cam were gifts of Drs. Karl DuS and I.C. Gunsalus of the University of Illi- nois, Urbana, Illinois. Cytochrome P450 partially purified from rabbit liver microsomes was a gift of Dr. M.J. Coon of the University of Michigan, Ann Arbor, Michigan. Samples of cytochrome P450 from rat lung microsomes were provided by Mr. John Buege. PB and 3—MC Pretreatment of Rats Rats were pretreated with either phenobarbital or 3-methylcholanthrene. Rats weighing between 200-250 g were pretreated by including 0.1%»PB in their drinking water 10 days prior to sacrifice. Rats weighing between 75-100 g; were injected i.p. with 3-MC (20 mg/kg in corn 15 oil) 36 and 24 hours prior to sacrifice. Liver microsomes prepared from each will be referred to as PB or 3-MC micro- somes 0 Preparation of Microsomal Membranes Rats were fasted 18 hours before killing by decapita- tion. The liver was extracted and the total microsomal fraction was isolated by differential centrifugation as previously described(34). The membranes were stored by suSpension in 0.05.§.Tris-HC1, pH 7.5, containing 50% glycerol to a protein concentration of approximately 50 mg/ml and frozen at -15°C under nitrogen in the presence of o.or% BHT. All isolation and washing procedures were carried out at 00-40C. Partial Purification of Cytochrome P450 and P448 The isolation procedure described by Levin,.g§.gl.(35) and modified by Welton(32) was followed. The final solu- tions, containing 3-8 mg/ml of microsomal protein, were frozen at -15°C under nitrogen. Immunological Techniques Preparation of Antibody Antibody was raised against cytochrome P450 (hemopro- tein 3) in rabbits by Dr. A.F. welton(32). Adult male 16 rabbits were immunized with three injections of hemoprotein 3 (3 mg protein/injection), administered in 2 ml of 50% Freund's complete adjuvant at one week intervals. Multiple intradermal injections of 0.1 ml were made into the abdomen. Ten days after the last injection, blood was collected from the marginal ear vein and serum separated from whole blood by allowing the blood to clot at room temperature for 3—4 hours. The clotted blood was placed at 0°-4°C overnight to constrict the clot; the blood was then centrifuged at 10,000 x g for 20 minutes at room temperature. The whole sera was removed by decantation and the IgG fraction iso- lated. The 196 fraction from both immune and pre—immune serum was prepared by ammonium sulfate fractionation and DEAE chromatography(36). Serum was centrifuged in a Sorval RC-ZB centrifuge at 40C for 20 minutes at 34,800 x g. The supernatant was decanted and the pH adjusted to 7.8. Ten ml of saturated ammonium sulfate (76.6 g in 100 ml) was added to 20 ml of supernatant at room temperature, stirred for 20 minutes, and centrifuged in an International Cen- trifuge at 3000 x g for 20 minutes. The pellet was resus- pended in 20 ml of 0.85% sodium chloride and the pH adjusted to 7.8. The ammonium sulfate fractionation was performed 17 again. The final pellet was dissolved in 15 ml of 0-015.H sodium phosphate buffer, pH 7.8 and dialyzed overnight against the same buffer. This partially purified IgG was run over a DEAE column (1 g DEAE per 50 mg protein) pre- equilibrated with 0.015 M.sodium phosphate buffer, pH 7.8. The 196 fraction came through in the void (Figure 2). Fractions were pooled and stored at 4°C.' Purity was deter- mined by SDS gel electrophoresis. Fab fragments were prepared from purified IgG(37,38). Forty-five mg of lyophilized IgG was dissolved in 3.5 ml of 0.1 M.sodium phOSPhate buffer, pH 7.0, containing 0.01 M cysteine and 0.002 M EDTA. Twenty v.1 of mercuripapain (25 mg/ml) was added and the mixture incubated for 16 hours at 37°C in a Dubanoff metabolic shaker. The solution was dialyzed against water and then against 0.01 §.acetate buffer, pH 5.5, overnight at 0°-4°C. After centrifuging the solution in an International Centrifuge at 3000 x g for 10 minutes, the supernatant was applied to a carboxy- methyl cellulose column and eluted with a 0.01.M.to 0.9 M acetate buffer, pH 5.5, gradient. The fractions were ana- lyzed by measuring their optical density at 280 nm (Fig- ure 3). The Fab fragments were eluted in two major protein peaks. 18 .HE H.m owcflmucoo sofluomnm comm .cESHoo may :0 mwfluflusmfiw umsuo pom swfisnam mcfl>mwa .mESao> UH0> may ca omusam cofluomnm 00H 039 .04. mm .3232.“ 53.08 m 30.0 a»? 0330 0cm 9:300 mean m 00 00300... mm3 m Samuoumoswsuwucm mcflcflmucoo Eamon Eouw OOH pmfimwusm SHHmHuumm mmOflDAflmu m¢WQ_20mh 00H mZDZSH m0 mQHmomm ZOHBDQH .m musmflm 19 ow Om .m canvas .02 20:041.... ON 0. S \lzli. m UJU 08200 N. Figure 3. 20 ELUTION PROFILE OF FAB FRAGMENTS FROM CARBOXYMETHYL CELLULOSE Fab fragments were prepared from purified IgG as described under Materials and Methods. The degraded IgG solution containing Fab and PC fragments was applied to a carboxymethyl cellulose column and eluted with a 0.01 M to 0.9 M.acetate buffer, pH 5.5, linear gradient. Four ml fractions were collected. The first peak contains Fab fragments and the second peak contains Fab and Fc fragments. 21 20 40 60 FRACTION NO. Figure 3. 22 Ouchterlony Double Diffusion Analysis Ouchterlony double diffusion analyses were performed as described by‘Welton(32). Thirteen ml of 1%.agar dis— solved in 0.015 M sodium phosphate, pH 7.8, 0.1% sodium deoxycholate, and 0.02% sodium azide were added to a dis- posable plastic petri dish (100 x 15 mm). The well pat- tern was cut, and antigen and antibody were added to appro- priate wells. Plates were developed at room temperature for 24 hours. Inhibition of Aminopyrine Demethylation Activity with Anti-Hemoprotein 3 N-demethylase activity was assayed by determining formaldehyde production(39,40). Reaction mixtures were incubated at 37°C under air in a Dubanoff metabolic shaker and contained microsomes (PB microsomes, 0.75 mg/ml or 3-MC microsomes, 1.25 mg/ml); 20 my aminopyrine: 0.05.§ Tris-HCl buffer, pH 7.5, to a final volume of 5 ml: NADP- isocitrate dehydrogenase (0.10 units/ml): 0.4 m1 of Solu- tion K (433 mg MgC12-6H20; 155 mg isocitric acid, trisodium salt: 25 mg NADP in 0.05 §.Tris-HC1 buffer, pH 7.5) and appropriate concentrations of antibody, either as 196 or Fab fragments. All but Solution K were pre-incubated on ice 3 minutes. After the addition of Solution K, the 23 mixtures were incubated at 37°C. At 3 minute intervals, 0.5 or 1.0 ml aliquots were removed and mixed with 0.5 or 1.0 m1 of 10%.TCA to precipitate protein. At the end of 10 minutes, 1.0 or 2.0 ml of NASH reagent (2 M.ammonium acetate, 0.05 M acetic acid and 0.02 M acetylacetone; diluted to 1 liter with water) were added and samples incubated at 60°C for 1 hour. Samples were centrifuged and the optical density at 412 nm was measured on a Perkin- Elmer model 124 spectrophotometer. Agglutination Assays The agglutination studies were conducted by an adap- tion of the methods described by Kwapinski(4l). Each assay (1.5 ml total volume) contained 0.14 mg of micro- somal protein, varying amounts of IgG and 0.85% sodium chloride. This mixture was incubated at 37°C for 5 hours and then centrifuged at 3000 x g for 10 minutes. The resulting pellets were washed once in 0.5 ml of 0.85% sodium chloride and then resuspended in 0.85% sodium chlo- ride for protein assays. As a control, microsomes incubated without antibody were also carried through this procedure, and the amount of protein found in the resulting pellet was subtracted from the amount pelleted in the presence of antibody. Incubation with pre-immune IgG gave the same results as controls. 24 The agglutination assays were also performed with iodinated microsomal protein. Iodination of microsomes was performed as described by Welton(32,42): pellets were diluted with cold microsomes and used in the assays. Complement Fixation Assays Complement fixation assays were used to test various antigen-antibody systems. The procedures outlined by Levine(43) and Kabat and Mayer(44) were followed. Reagents: Stock diluent - To 800 ml of distilled water were added 81.6 g of NaCl, 12.1 g of Tris base, 6.6 ml of concentrated HCl, 33 ml of 0.15IM.MgSO4, and 15 m1 of 0.1 M.CaC12. The solution was mixed, the volume adjusted to 1 liter, and the pH adjusted to 7.4. This stock buffer was diluted 1 in 10 just before use and 0.1% bovine serum albumin was added. Alsever's Solution - To 1200 ml of distilled water were added 24.6 g of glucose, 9.6 g of sodium citrate (dihydrate), and 5.04 g of NaCl. The pH was adjusted to 6.1 with citric acid, and the solution was sterilized by passing it through an ultrafine Sintered glass filter. Sheep Red Blood Cells - Sheep red blood cells (50 ml) were drawn aseptically into 70 m1 of Alsever's solution and stored at 2°—5°C for one week prior to use. Four ml SRBC 25 were centrifuged at 4300 x g for 10 minutes; the cells were then washed 3 times by suspending the cells in 1.0 ml of diluent and centrifuging at 4300 x g for 10 minutes. After the last wash, cells were resusPended in 18.0 ml of diluent and filtered through glass wool. To determine cell concentration, 1.0 ml of suSpended SRBC was lysed with 14.0 ml of 0.1%1Na2C03 and the optical density at 541 nm measured. Adjustments were made until an CD of 0.680 541 was Obtained. This corre8ponds to a RBC concentration of l x 109 cells per m1. Hemolysin — Hemolysin was reconstituted, then diluted 1 in 50 with diluent and stored at -20°C. For use, 10 ml of hemolysin, diluted 1 in 1000, was added slowly and with constant swirling to 10 ml of suspended SRBC, incubated at 37°C for 15 minutes and diluted with 180 ml of diluent. The sensitized cells were prepared daily and stored at 4°C. Complement - Complement was reconstituted and stored as 2 m1 aliquots in screw cap tubes at -20°C. Once restored, the complement was good for 2-4 weeks. Titration of Complement: To determine the optimum concentration of complement to be used in assays, comple- ment was titered. One ml of complement, diluted in the range of 1/100 to 1/250, was mixed with 5.5 m1 of diluent in a 40 ml Sorval centrifuge tube and incubated at 0°-4°C 26 for 16—18 hours. The samples were then incubated with 1.0 m1 of sensitized SRBC for 60 minutes at 37°C in a Dubanoff metabolic Shaker. At the end of the incubation period, samples were cooled on ice to stop the reaction and centri- fuged at 4300 x g for 10 minutes. The supernatants were measured at 413 nm. The percent hemolysis for each sample was calculated and plotted against complement dilution. The highest dilution giving 90% hemolysis was used in fu- ture assays (Figure 4). Titration of Hemolysin: Hemolysin (antibody to sheep red blood cells) was serially diluted from 1/400 to 1/6400. Five ml of hemolysin was added to 5.0 ml of washed SRBC with constant swirling: thus, for each hemolysin diluted, cells were sensitized. Each assay (total volume, 6.5 ml) contained diluent, 1.0 m1 of complement appropriately diluted, and 1.0 ml of sensitized SRBC. The samples were incubated at 37°C for 60 minutes, cooled on ice and cen— trifuged at 4300 x g for 10 minutes; optical densities at 541 nm of the supernatant were measured. Percent hemolysis was calculated and plotted against hemolysin dilution (Figure 5). The largest dilution giving 50% hemolysis was used. Titration of Antibody: To determine the concentration of antibody to be used and the range of antibody—antigen 27 .SOMumuuwu was» Eoum mocHEumuop 003 oom\H mo coHuSHam « .mhmmmm cofiumxflm ucmeamEoo ouSuSm a“ mom: mm3 mamhaosmc.*om mcfl>wm ucmfiwamfioo mam mocfism mo cofluSHHo umomuma one .moonumz 0cm maneuoumz woos: omnfluommp mm >mmmm cofiumxwm ucmEmHmEoo may cw pmumuuflu mm3 ucmEonEoo mwm mwcfisw BZHZWQQZOO OHm flmZHDw ho ZOHEmmBHB .v musmflm 28 COS .v musmflm 20:51.5 FZMZMJQZOO OON\. .0. SISA‘IOWBH % 29 .oooa\a mo coflusaflo m on popcommmuuou mace .mammmm ceapmxflw ucmsoamEoo ucmsummnsm ca poms mm3 mflm>HOEmc Row mcfl>Hm coHuSHflo ummmuma one .moonuoz 0cm mamwuwumz Hops: confluummo mm omumufiu mm3 cflmhaosmm ZHmMQQSME m0 ZOHBmMBHB om musmflm 30 .m musmflm 20:54.0 z_m>._02m1 00¢: 00m) 5 d u SISA'IOWBH‘X: 31 interaction, antibody was serially diluted from 1/200 to 1/6400 and antigen from 0.01 to 10 ”g of protein. To each 40 m1 Sorval centrifuge tube (total volume, 6.5 ml) was added: 1.0 ml of appropriately diluted antibody, diluent, 1.0 m1 of complement, and 1.0 ml of antigen. The tubes were vortexed and incubated at 0°-4°C for 16-18 hours. One ml of sensitized SRBC was added and samples incubated at 37°C for 60 minutes. At the end of the incubation, samples were cooled on ice and centrifuged at 4300 x g for 10 minutes. Supernatants were measured at 413 nm and the percent of complement fixed was determined. In all assays four controls were included: 1) diluent and antibody: 2) diluent and antigen: 3) diluent and complement: and 4) diluent only. The highest antibody dilution giving a maximum fixation of 70% was used (Figure 6). Complement Fixation Assay: The complement assay was performed by adding, in order, to a 40 ml Sorval centrifuge tube: 1.0 m1 of antibody, diluent, 1.0 ml of complement, and 1.0 m1 of antigen. The four controls described under "Titration of Antibody" were included. After samples had incubated at 0°-4°C for 16-18 hours, 1.0 m1 of sensitized SRBC was added. The tubes were then incubated at 37°C for 60 minutes, cooled on ice, and centrifuged at 4300 x g for 10 minutes. Optical densities at 413 nm of the supernatants 32 .31: 0mm: £<|S 00m) 2...! 0mm: £0|9 00m: “98336 6335 .m 53300523000 you .8336 00m) 0 Op popcommwuuoo mane .mwmmmm nodumxwm ucmEmHmEou ucosvmeSm ca can: 003 poxwm ucosmameoo Ron mo EsEame m mca>am coHuSHHp unwound one .moosumz 0cm mamfluwumz Home: omnwuomoo 003 m Seououmofioclwucm mo coHumuuwu use m ZHmBOMmQmeIHBZ¢ m0 ZCHBmMBHB .0 wusmfim 33 0.. .0 ousmfim on .Z_w...omn_ szomomgz QC v.0 O BXL-J 1N3W3'IOWOO 96 00. 34 were measured and used to calculate the percent of comple— ment fixed. A typical protocol is given in Table I. SDS Polyacrylamide Gel Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electropho- resis was performed as described by Fairbanks(45) and modi- fied by Welton(32). Gels were scanned at 550 nm in a Gil- ford spectrophotometer equipped with a gel scanning attach- ment. Other Assays Protein concentrations were determined by the method of Lowry(46). Cytochromes P450 and P420 were assayed as described by Imai and Sato(47). A computer program was used to calculate the concentrations: extinction coeffi- cients of 11 and -11 cmflmM‘l‘between 450 nm and 490 nm for P450 and P420, respectively, and -41 and 110 cm'lmM"1 'between 420 nm and 490 nm for P450 and P420, respectively, were assumed. 35 Houucooao 0 un \oOOH MHvQOQ umBOHHOM mm omc«Eumumo ma omxam ucosmamsoo unmoumm was .Houucoo may scum moamsmm on» no >ufimcoo Havaumo on» mcwuomuunSm an omumasoamo ma huwmcwo Hmoflumo CH wmcmno one .maouucoo usom evades“ mammmm mo mmwumm sumo can moaned smouo on» a“ 00000 mum mucocomsoo .30Hmn cw>fim mum mammmm coflumxfiw ucoswamsoo now Honououm Hmofimxu m mwdmmm ZOHB¢XHh BZHZWQASOU mom ROUGEomm .H OHQMB 36 00000 00000 000 @0000 00u0u0ucuu 1 cm 0008009800 | .O 000: 030000 00003.8 .. .2 000.0 0.0 II: III 0.0 II: 00 000.0 0.0 an: 0.0 0.0 In: 00 000.0 0.0 In: 0.0 0.0 0.0 00 000.0 0.0 0.0 0.0 0.0 III 00 0 000.0 000.0 0.0 0.0 0.0 0.0 0.0 00 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 00 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 000 000.0 000.0 0.0 0.0 0.0 0.0 0.0 m 000 000.0 000.0 0.0 0.0 0.0 0.0 0.0 0 09350 x m0000 «.0000 0s 0s 0s 0s 0a .02 .00 .com0uc4 ..U .ucma00n .00 mass H 00309 RESULTS An antibody directed against hemoprotein 3, the 44,000 dalton microsomal protein which is induced in rats by pre- treatment with PB, was prepared and used in these studies. Since the antibody appeared to have a low titre (1/300 dilution = 4.7 '1g IgG) as assayed by complement fixation, two other cytochrome P450 antigens were injected into rab- bits in attempts to elicit better antibody reaponses. _The first antigen was the degradative form of hemoprotein (cytochrome P420) in which the solubilizing SDS reagent had been removed. The second antigen was composed of hemo- protein 3 crosslinked to rabbit serum.albumin(48,49). Neither antigen elicited an antibody response as tested in Ouchterlony double diffusion with solubilized whole microsomes. In either case the structure of the antigen may have been drastically altered so that the antibody no longer recognized the hemoprotein found in microsomes or the antibody titre was extremely low. Thus, the original antibody to hemOprotein 3 was used and will be referred to as anti-hemoprotein 3. 37 38 The two major components present in all systems were microsomes from various sources containing cytochrome P450 and the antibody to hemoprotein 3. SDS gel electrophoresis was performed on all proteins used. The gel scans, shown in Figures 7 and 8, refer to whole microsomes from PB, 3-MC, and control rats: partially purified cytochrome P450 from microsomes of PB, 3-MC, and control rats: rat lung microsomes: cytochrome P450cam from.g. putida: partially purified cytochrome P450 from rabbit liver microsomes: hem0protein 3 (antigen): and IgG containing anti—hemopro- tein 3. Ouchterlony double diffusion analyses were performed to determine the degree of cross-reactivity of the antibody with various sources of cytochrome P450. In Figure 9, a precipitin line was Observed only with microsomes from PB—pretreated rats: no cross-reaction occurred with P450cam, rabbit liver P450, or rat lung microsomes. In Figures 10 and 11 precipitin lines were Observed with PB, 3-MC, and control microsomes and with partially purified cytochrome :P450 from PB, 3-MC, and control microsomes. No Spurs were (Observed, indicating common antigenic sites. There was no observable reaction of pre-immune IgG with any of the micro— somes . 39 .0u0u 0000muuonmnuzu0 000 :00 080 0000000 8000 00000000 0080000008 8000 00000050 >0000uumm 0000 0800500000 080000900 08000 00300 009 .0000 pounmuuoumluzum 000 I00 000 0000000 8000 00u00000 0080000008 00 000 00000 woman was mfimm mafifimmafimmIUZIM 92¢ Imm nzc AOfiBZOU 20mm GmfidflOmH mm20m0m0H2.20mm QflHhHmDm NAHflHBmmm momwm mzommUOBwu mmfi QZ£ mafim Dmafimmfimmmluzlm 92¢ Imm 02m HomBZOU 20mm QHBGAOmH mmZOmomUHz m0 mMAHmomm ZHmsomm mHmmmommomBUHAm HMO mnHzaflhmvflwfiomlmnw.XH .0 00500h 40 02h .b 000m0m Eu 824.55 29.25.! 0 m c N 10.1)014 j -O 7 . 41 .000000000V m 00000000800|0000 000 000m0000v m 00000000800 00 000 00000 00300 000 .0080000008 00>00 009000 000 .00000m oM..0080000008 @000 000 ”000000 00 0000v 8000 omvm 0800000000 00 000 00000 00000 009 m 00000000000u0000 000 m 00000000000 000 00000 -0000: mm>00 000000 000 .000000 00000000000 .0000000000 0000 000 0000 0000 0200000000 00 00000000 0000000 000000000000000 000 00000000000000-000 00 .m 000m0m m m ¢ N 0 300 2 nthocg..- . _ 47/5 59¢ 0.0 £024.55 29.25.: «Gr—bum 3 4 .0000>00000000 .mi hm 000 mi. mmv 0000 5000 0000000 0 000 m 00003 .004. 00v 0050000005 00>00 000000 5000 00000000 0 0003 .001 mvv 0000 0000000000100 5000 00000000 omv0 05000000>0 00000000 m 0003 .AmL mmv 5000m¢0 0500000000 0050000005 0000 0000 0500000000 0050000005 5000 00000000 N 0003 .m.h 00 .000000000 500000 m.m0o.o 00000000 0 0003‘ .00 .020 000 000 5000 0003 100000 00000500 000 000 00000000 000000> 0000000 00003 00000 000 .00000000 00 mi 0000 m 00000000500u0000 00000000 0003 000000 009 .0000002 000 000000002 00000 000000000 00 005000000 0003 00000000 000000000 000000 00000000000 mmUMDOm AdzOm IOmUHZ mDOHm€>.SOmm QflBfiQOmH omfim mzommUOBVU QZ¢ m ZHmsommozmm OB MGOflHBZfl 2mm39mm mZOHBU¢mm ZHBHmHUmmm mmfi 00 mmeAQZQ ZOHmDhmHQ MANDOQ_WZOAmmBEUDO .0 000000 44 Figure 9. 45 .001 vmv 0000 0000000 5000 0050000005 0000000 0 000 v 00003 .001 ovv 0000 0000000000u021m 5000 0050000005 00000000 m 0003 .00000 04. 000 0000 0000000000|00 5000 0050000005 0000000 m 000 N 00003 .m.h 00 .000000000 500000 m.m0o.o 00000000 0 0003 .00 .020 000 000 5000 000300000 00000500 000 000 00000000 000000> 0000000 00003 00000 009 .00000000 00 04. owwv m 0000000050sn0000 00000000 0003 000000 009 .0000002 000 000000002 00000 000000000 00 005000000 0003 00000000 000000000 000000 00000000000 mBmm QMB4MMBmm0IUSIm QZ€ Imm Q20 0008200 20mm QmBmAOmH mMZOmomUHZ QZ¢ m ZHmeommozmm OB MDCflHBZfl me39mm mZOHevdmm ZHBHmHUmmm HEB mo mmmfifimzm ZOHmDhmHQ mAmDOQ_WZCQMMBEUDO .00 000000 46 Figure 10. 47 .004— mmv 0050000005 0000000 5000 00000000 000000000 om¢0 0500000000 0000000 0 000 0 00003 .001 may 0000 0000000000|Uz|m 5000 00000000 0050000005 5000 00000000 000000000 0000 050000 :0000 00000000 m 0003 .000. mmv 0000 0000000000|00 5000 00000000 0050000005 5000 00000000 000000000 om00 0500000000 0000000 m 000 N 00003 .m.b 00 .000000000 500000 m.m0o.o 00000000 0 0003 .00 .ozv 000 000 5000 000300000 00000500 000 000 00000000 0000000 0000000 00003 00000 008 .00000000 00 mi owmv m 00000000500I0000 00000000 0003 000000 009 .0000002 000 00000000: 00000 000000000 00 005000000 0003 00000000 000000000 000000 00000000000 mafim Quammmfim00IQZIm QZG I00 020 0009200 20mm DEBflncmH mmZOmOMUHZ £000 QMHmHmDA MnamHBm<0 omvm MSOKEUOBNU 02m m 2HmfiomeSmmlHEZ¢ ZumSBmm mZOHfiummm ZHBHmHUmmm HEB 00 mmmNAmZ¢ ZOHmDhmHQ NAMDOQ MZOQMHBEUDO .00 000000 48 Figure 11. 49 The inhibition of aminopyrine—N—demethylation in micro- somes isolated from PB and 3-MC-pretreated rats with puri— fied 196 to hemoprotein 3 is shown in Figure 12. The reacé tion was not inhibited, even at IgG to microsomal protein ratios of 4.5:1 and 3.1:1 for microsomes isolated from PB- and 3-MC-pretreated rats, respectively. Microsomes also agglutinated at the higher antibody to protein ratios, indicating that the antigenic sites were located on the exterior surface of the microsomal membranes and were not interfering with the enzymatic sites. To avoid the pr0blem of agglutination, Fab fragments instead of IgG were used in the assay. The results, given in Figure 13, show only slight inhibition of aminopyrine— N—demethylation in microsomes isolated from PB-pretreated rats at a Fab to protein ratio of 11.6:1. Thus, the inhibi- tion of aminopyrine-N—demethylation in microsomes from PB— or 3-MC-pretreated rats with immune IgG or Fab fragments was unsuccessful. The enzymatic sites may be, therefore, buried within the microsomal membrane or are on the exterior surface of the membrane and not affected by the binding of antibody. Agglutination studies were performed to determine if anti—hemoprotein 3 would preferentially precipitate micro- somes from PB-pretreated rats. Differences were observed O 5 .00....3 000 0o 0.: 0.00 .8 .Afllfi 000 0o 0.: m.m 20...! 000 00 02 0.0 .303 050 3'0. 00000000 030003 0000 0000000000|Uz|m 5000 0000000 0050000005 00 m5 0.0 00000000 0 00 00000X05 0000 -0000 0:0 .Aou.0. 000 00 02 0.00 00 .Aaqug 000 0o 00 m.m .001100 000 00 05 0.0 0003 000 AOIIOV 000 000550 0000003 0000 0000000000Im0 5000 0000000 0050000005 00 05 mb.m 00000000 0 000 .0000002 000 000000002 00000 000000000 .00000005 00000000 009 .0000000000 000000005000 00000500000 00 0000000 003 m 0000000050010000 0003 0000000000500Iz|00000000050 00 000000000H 60H WZQZSH EBHB mfimm QMB¢HMBflm0IUSIm 92¢ Imm 20mm QQBQQOmH mmSOmomUHZ 2H 20HBmflwmfimzmQIZIHZHMNQOZHZQ mo ZOHBHmHEZH .00 000000 .00 000000 mwhaz=2 Us...— 0 N. 52 .AIIIIV nucoemmuu Dam 0:555“ m0 m8 ma no “ATLQQ mucoEmuum nah 0:585“ noun no 08 OH cuw3 van AOIIOV anonwpco usosuAS mumu nmumouuonmlmm Scum cfiwuoum anaemouowa no me mm.H omosHOCw .mGOLumz can uHMfiuwumz noon: cmfifinomoc .mousuxAE newuommu one .cofluosooum mohaocamfiuou an omammmm «M3 mucofimmum nah m admuOHQOEmsnfiuCn cuwz so“umaznumEmQIZuocfluhmocfiEm mo cowuanfincH mBzmzwmmh mflm HZDZZH 92¢ mZDZZHImmm EBHS mfifim nmafimmBHMA Imm 20mm QmB¢AOmH meOmOmoHZ ZH 20HBdAMmBMZMQIZIMZHMMmOZHzc m0 ZOHBHQHMZH .mH musmflm 53 .mH musmfim mm...32.2 .w_2_._. vN m. N. fi d J d 0_.O ONO ”J“ Zlbgo Ono 54 in agglutination of microsomes from control and PB- and 3-MC-pretreated rats (Figure 14). If the 44,000 dalton cytochrome P450 is induced in microsomes from PB—pretreated rats and is present in higher levels than in microsomes from control or 3-MC-pretreated rats, then one would expect microsomes from PB—pretreated rats to agglutinate more readily than the other two. Such a relationship was Observed. Microsomes from PB-pretreated rats agglutinated three times as much as microsomes from control rats; micro- somes from 3-MC-pretreated rats precipitated twice as much as microsomes from control rats. Attempts to assay the degree of agglutination with iodinated microsomes were unsuccessful. The differences that were Observed in Figure 14 are missing in Figure 15. If hemoprotein 3 is induced in PB-pretreated rats and is, therefore, present in greater quantities than in control microsomes, then the amount of labelled hemoprotein 3 in PB microsomes should be greater than the amount of labelled hemoprotein 3 in control microsomes. As a result more labelled microsomal protein from PB-pretreated rats should be agglutinated than labelled protein from control rats. The Observation that equal amounts of labelled microsomal protein from either control or PB-pretreated rats implies that iodinated microsomal protein is preferentially .AOIIOV numu Umuwouuwumlmm can AlTilv omummuumumluzlm Eouu nonmaomw noEOOOHOwE uo>fla mo umcu ou omummeoo mw3 adrlqv mmEomouoflE Houucoo mo OOwumcwusHmmm one .moonumz can mamfluoumz nouns confluomoo no vmhmmmm mm3 mumu noumouuoum IUZIm can 1mm was Houucoo Eouw omumaomfl noEomouoflE mo COwOMCausHmmm 5 5 m ZHMBOMAQZHEIHBZ¢_mBH3 mama Gmaflmma ImmmIUZIm Dzm Imm 92¢ HomBZOU 20mm QMBQAOmH mMZOmOMUHS ho ZOHB¢ZHBDQUO¢ o¢a musmah 56 l80- '5 o PRECIPITATED PROTEIN, pg G O N O l l l I l L LO 2.0 3.0 IgG ADDED, mo Figure 14. 7 5 . 3'9 33 noumouuoumumm Scum coumHOOfl noEomouofiE mo away on woummeoo nm3.Aerqv mmaon souofis owaaonmauumma Houucoo mo COwumcwusammm one .mponuoz 0cm mamwuoumz noon: confluomop mm oowmmmm muo3 Hmma nuw3 omumcfloofl 0cm mama omumonumum noz:m can Inc can Houucoo Eouw ooumHOmd moEomouOfiE mo cofiumcflusammm m 2HmBOmemeIHB2¢.mBH3 m9¢m 9NB¢mmfimmm IUEIm 92¢ I99 92¢ QOmB2OU 2029 9MB¢90mH mmzowomUHZIHmNH mo 20HB¢2HBDQUG¢ .ma magmas 58 40.000l- 30,000 20.000 no,ooo ' '25I-PRECIPITATED PROTEIN, cpm L0 20 3.0 IgG ADDED, mg Figure 15. 59 agglutinated. 0n the basis of the original protein assay which quantitatively determined the amount of agglutinated protein, differential agglutination by microsomes from control and PB— and 3-MC-pretreated rats was Observed. Complement fixation assays were performed with various microsomal sources to demonstrate the specificity of anti- hemoprotein to hem0protein 3. In experiments using whole microsomes from control or PB- and 3—MC-pretreated rats, differences were Observed in the amount of complement fixed (Figure 16), thus implying differences in the amount of antibody bound by each. If more antibody bound to liver microsomes from PB-pretreated rats than to control or 3-MC- pretreated rats, then the antibody would appear to be spe- cific for the hemoprotein induced by pretreatment with PB. This effect was Observed. At low concentrations of micro- somal protein (0.8 .Lg), it is possible to quantitate the relative amount of antibody bound to the three types of microsomes. Assuming that the amount of complement fixed is directly pr0portional to the amount of antibody bound, it appears that microsomes from PB—pretreated rats bind approximately three times the amount of antibody as do control microsomes while microsomes from 3-MC-pretreated rats bind twice as much as do control microsomes. 60 .omH 0:525“ 90 mi. h.¢ nonamucoo >mnnm comm .AIIIIV mums nouoouuoumlozlm 0cm AdeQg Houucoo Eoum nonmaoma ooEom lauofie nuw3 codumxwm mo ucsoEo ozu ou nouwmeoo nm3 AOIIOV mumu coummuuoum 1mm Eoum noumHOmH noEomoquE nuw3 ucoanmEoo mo coflumxam one .noonumz can manfiuwumz noon: confiuomoo no coauowuom oum3 nkonnm COADOxam ucofioamfiou mmSOmomUHz 99023.28H3 m ZHWBOmmOZWmIH82¢ mo mmAHmomm 20HB¢NHm BZNZMQQZOU .oH magmas 61 9.? O.¢ .OH magmas a... . 2.5.01.“ 4 ocm omH 0:988“ 00 m1 h.v posamucoo woman zoom .noonuoz can mamwuoumz moons confluonoo no coauowuwm mums nhmnnm coflumxwm ucoEmHmEoo m 2H990290292IH92¢ mBH3_m920m020H2 0299 B¢m 90 MQHhomm 20HB¢NHh 8292999200 .mH magmas 66 .ma magmas a; .2905 452808.: On ON 0. - q - u 0 N % O V O O (D (D OBXId lNBWEWdWOO 00. 67 those organs in rats, i.e., lung microsomes in rats contain much less cytochrome P450 than liver microsomes. Since lung microsomes from rats did bind antibody, the antigenic sites must be similar to those found in liver microsomes from rats. Cytochrome P450 partially purified from rabbit liver microsomes and cytochrome P450cam purified from g. putida were compared with cytochrome P450 in whole microsomes isolated from PB-pretreated rats. One can infer from Figure 19 that no antibody is bound to cytochrome P450 from rabbit liver microsomes or cytochrome P450cam within the protein range examined (up to ten y-g of protein). The antibody is specific for cytochrome P450 from rat liver microsomes. These results imply that few or no antigenic determinants for hemoprotein 3 are found on cytochrome P450 from rabbit liver microsomes or cytochrome P45°cama An antibody to cytochrome P450cam‘was tested in the complement fixation system with cytochrome P450camo cyto- ' chrome P450 from rabbit liver microsomes and cytochrome P450 in whole microsomes from PB-pretreated rats. As shown in Figure 20, complement was fixed by cytochrome P450cam but not by cytochrome P450 from rabbit or rat liver micro- somes° The antibody appears to be specific for cytochrome P450cam implying that no common antigenic determinants 68 .AIIIIV mmflmmm..M_Eouw Emoomem oEounoouho “AAPLQQ noEououofiE uo>99 ugnnmu Eouw omvm oEoun00u>o oofiuausm maaufiuummvaAOIlOv numu oouwouu noumlmm Eoum ooumaomfi omvm oEOusoouao Hmfiooouowfi «unwououm mo noodumuucoo Icoo modaum> pom 00H oases“ 90 m4. h.v oocflmucoo human comm .uoonuoz can mamfiuoumz noocs confluomoo mm ooEuomuom ouo3 masons nodumxam ucoEOHQEoo ¢9HBDA .9 92¢ m9209090HZ 99>H9 BH99¢9 92¢ 99>H9 B49 2099 99B¢909H omvm 9209900820 99H3 m 2H990990299IHB2¢ 90 m99H9099 20HB¢NH9 9292999200 .mH magmas 69 .ma ousmam a; .z.u._.omn_ m0 0.0 to «.0 1 d ‘ ¢ 0 N o o no 0 ('3de .LN3W 31dW00 70 a 9 70 .adrlqv upon counouuoumlmm Eoum ooumaoma noEOOOuoHE «Alla-v umEOOOquE uo>aa uannmu Eouu twamausm aaamwuumm omen oEoucOOuao “QOIIOV_Emoomvm oEounoouho .ocaououm mo nc09umuu=oocoo mcwmuo> can oom\9 omusafio Emoomqmlflucm cocwmucoo woman zoom .uoonuoz can mamauoumz woos: oonwuomoo no coauomuom ouo3 masons nodumxflu ucoEonEoo coHeom .m nza mmZOmomon ensue summam oza mused Ham 20mm omen mzommooewo meHz.2moomvm mzommooewouaeze so mmqnmomm oneeme ezmzmgmzoo .om ouawam '71 .om ousmam s. .2905 om o._ no , no: .23. nun-‘4 < 6L.-.II I CO ¢N O o co co GBXIJ .LN3W3'IdWOO % °\ o 9 72 exist among cytochrome P45°camo cytochrome P450 from rabbit liver microsomes and cytochrome P450 from rat liver micro- somes o DISCUSSION The results presented in this thesis are further evi— dence that anti-hemoprotein 3 is specific for hemoprotein 3 and that multiplicity of cytochrome P450 does exist. By using four different immunological techniques (Ouchterlony double diffusion, antibody inhibition of enzymatic assays, agglutination, and complement fixation) the experiments conducted have provided consistent results which support three ideas: 1) the antibody is specific for a particular antigen: 2) the same protein (hemoprotein 3) may be found in other organs of the same species: 3) the active site of cytochrome P450 appears to be buried within the microsomal membrane. The first line of evidence for specificity was provided by Ouchterlony double diffusion analyses. If the antibody to the 44,000 dalton rat liver microsomal cytochrome P450 were specific, then it should bind only to hemOprotein 3. This was Observed. The antibody reacted with those micro- somes that contained hemoprotein 3 (microsomes from control and PB- and 3-MC-pretreated rats) and failed to cross-react 73 74 with cytochrome P‘socamv rabbit liver cytochrome P450 and rat lung microsomes. The second and third lines of evidence were Observed in the agglutination and complement fixation studies of whole microsomes from control and PB- and 3-MC-pretreated rats. Since hemoprotein 3 is induced in PB—pretreated rats and the antibody has been raised against this protein, one would expect more antibody to bind to microsomes from PB- pretreated rats. As more antibody was bound, more aggluti- nation would occur and more complement would be fixed. By using results in each assay to quantitate the relative amount of antibody bound to the three types of microsomes, one Observes that liver microsomes from PB—pretreated rats bound three times as much antibody as did microsomes from control rats and microsomes from 3-MC-pretreated rats bound twice as much antibody as did microsomes from control rats (Table II). It is difficult to quantitate the concentration of the 44,000 dalton hemoprotein found in each type of micro- some to make relative comparisons, but a rough estimate can be Obtained by comparing 1%.SDS-polyacrylamide gel electrophoresis protein profiles. Integration of peak areas can be made to quantitate the amount of hemoprotein 3. In terms of relative proportions, liver microsomes from .nOHuoo usHsOwuuom ans» :9 «099 name douucoo uom co>fim nonesc on» on uoomnou nua3 amass: nos» «0 coduuom Iona on» 09 uonEs: comm measoaaom monocucouom may cH .omvm oEouzoouho mo >ua>9uoo 02990090 on» nonwuueaso m casaoo .hao>fluoomuou .Omcm oeounoouho pawmwusm haaswuumm can uoEonouowe oHoc3 scum omen oEOucoouxo mo noaoec Mica x o.~ can -09 x m.o um OOXAM ucoEmamEoo mo assess on» oo>wm m GEOHOU .noafiwoum cwououm 90m mnm_$9 Eouu ccsow Am cwououQOEozv o comm No.8 on» oo>9m v canaoo .9Ho>wuoomnou .omvm oeounoouwo pawuausm xaasduumm can u” uoEouou09E oaon3 Eoum cwououm Hmeomouofie «0 ma. «.0 was 0.0 an maxim ucoE loamEoo mo ucsoam on» mo>am m cesaoo .m09csuu COwumcwusammo Eoum me no me m.~ um poumufimwooum awououm mo ucsoEm on» mo>wm N casaou .omvm oEoucoouho mo mouson on» mouocoo casaoo unnam one .uconoum m swououmosoc mo ucsofim on» on uoommou :u93 amaosum moodum> mo unannou onu mouaumEEan HH manna omvm 9209900920 90 MBH>H90¢ 0H9909mw 999 92¢ 9990 m9ml¥a 20 8299999 m 2H990990299 90 92902¢ 999 06 9099999 98H3_99H9DBm 20HB¢NH9 8292999200 92¢ 20HB¢2HBD900¢ 2099 9999999 90 >9¢22Dm .HH 09908 76 noes... 23338 x. u to x xo.He m.~ Ao.ae om lo.av e.ma .o.HV Hm uuuuuuu Houucoo ae.av m.v Ao.ov om Am.ov m.ma Am.ov pw nuuuuuu uzum an.~v m.m xv~.He no .~.HO m.- .m.~v «m nuuun-n mm omen «sensuouao omauauam meanauuam Ao.H. 5.0 Ao.H. ow xo.H. m.m Ao.H. en Ao.He Hm Houucoo .o.~e ¢.H .>.oe ma “p.90 o.m xm.ae he .o.av mm usum Ae.mv o.~ Am.o. on Am.~. m.ma .m.nv as A¢.«. vaa mm nofionouofla 090:3 cwououm we won. omwm mOHOEc zoom 900 :wououm Gwououm mamfiom omvm nOHoE: 9.0 x. mam Souk AMEowouoaz mousuamaooum 333an 333% m 38 x Fe x coauofiufimma IllIlIIlIlllIlIIIlllllIIlIlIlllIlllllIllllIlIlllllIIIIllIl|lllI|llllllllllllllllllllllll HH 099MB 77 PB-pretreated rats contained three times as much hemopro- tein 3 as did microsomes from control rats and microsomes from 3-MC-pretreated rats contained twice as much protein as did microsomes from control rats. The correspondence between the proportions Obtained with agglutination and complement fixation assays and with integration of peak areas on r% SDS-polyacrylamide gel protein profiles argues for the specificity of the antibody. The fourth line of evidence supporting antibody speci- ficity was Observed in complement fixation assays involving cytochrome P450 partially purified from three types of whole microsomes. The proportion of hemoprotein 3 found in each type of microsome can be roughly estimated by inte- gration of peak areas from T% SDS-polyacrylamide gel elec- trophoresis protein profiles. The ratios calculated for _1 cytochrome P450 from microsomes isolated from P3- and 3-MC- pretreated rats as compared to that from control rats were 1.2 and 0.8, respectively. A similar relationship was observed with respect to the amount of antibody bound in complement fixation assays except that cytochrome P450 from PB-induced rats bound 2.5 times as much antibody as did cytochrome P450 from control rats. The discrepancy ob— served with respect to the relative amount of antibody bound by cytochrome P450 from PB-induced rats compared to 78 that from control rats, i.e., 1.2 and 2.5, may be an idio- syncrasy of the purification process of cytochrome P450. More antigenic sites per molecule of cytochrome P450 from PB-induced rats may be exposed during purification. If the mixed-function oxidase system is found in other organs of the same species and contains the same protein components, then one may hypothesize that the systems also have a similar hemoprotein 3. Rat lung microsomes contain cytochrome P450, but in much smaller quantities than is found in rat liver microsomes. Given that the antibody is specific for hemoprotein 3, the antibody can be used in the complement fixation assay with rat lung microsomes to ascer- tain if hemoprotein 3 is present in rat lung microsomes. It was found that rat lung microsomes fixed complement when at least ten times the amount of rat liver microsomal protein was present. These results imply that similar anti- genic sites for anti-hemoprotein 3 exist in the cytochrome P4508 isolated from various organs within the same species. Additional evidence that anti-hemoprotein 3 is specific for hemoprotein 3 from rat microsomes was suggested from results of complement fixation with cytochrome P450 and cytochrome P450cam isolated from rabbit liver microsomes and g. putida, respectively. No complement was fixed by either cytochrome in the presence of anti-hemoprotein 3. 79 These results indicate the lack of similar antigenic deter- minants among cytochrome P45°camo rabbit liver cytochrome P450 and rat liver cytochrome P450. Anti-hemoprotein 3 is specific for rat microsomal hemoprotein 3, which appar— ently is not present in the other two systems. The antibody to cytochrome P450cam was also tested by complement fixation with cytochrome P45°camt rabbit liver cytochrome P450, and rat liver cytochrome P450. Only the system containing cytochrome P450camganti-cytochrome P45°cam fixed complement: therefore, there appear to be no common antigenic determinants among cytochrome P45°camc rabbit liver cytochrome P450, and rat liver cytochrome P450. The results with the anti-cytochrome P450cam and anti- hemoprotein 3 were quite surprising since recent experiments performed in Coon's laboratory(26) were contrary to what a“ we expected. In their reconstituted system, the hydroxyla— tion of benzphetamine in the presence of purified cytochrome P450 isolated from rabbit liver microsomes was inhibited by antibodies to rabbit liver cytochrome P450 and to cytochrome P450cam- Both antibodies must have similar antigenic determinants in order to cross-react with rabbit liver cyto- chrome P450. This discrepancy indicates the necessity to test their antibodies and antigens in our complement fixa- tion assay. If complement is fixed only for specific 80 antigen-antibody reactions, then the inhibition of benz- phetamine hydroxylation by both antibodies is rather dubious. If, on the other hand, cross-reactions do occur, then there are distinct differences between our antibody to rat liver hemoprotein 3 and their antibodies. It is inconceivable, though, that the same antibody to cytochrome P450cam would not fix complement with rabbit liver cyto- chrome P450 but would inhibit an enzymatic reaction. Returning to the initial complement fixation studies discussed above, one can Observe an interesting problem when the amount of complement fixed is plotted against the concentration of cytochrome P450 (Figures 21 and 22). In Figure 21, the relationships among cytochrome P450 isolated from control and PB- and 3-MC-induced rats are no longer 3:2:1 for cytochrome P450 isolated from PB, 3-MC, and control microsomes, respectively. At 0.8 x 10‘3 nmoles of cytochrome P450, it appears that cytochrome P450 from control microsomes binds twice as much antibody as gx .\ . s 7 ’ does cytochrome P450 isolated from PB microsomes. If, in terms of microsomal protein, three times as much antibody was bound by microsomes from PB-induced rats than from control rats, than it is not logical that expressing the amount of antibody bound in terms of nmoles of cytochrome P450 would alter the relationship between the amount of 81 . 23 «you Houucoo Eouu nosonouofle 090:3 5 035 0805200qu 2.1.! open nouoouuoum nozum Eouu noeououofle 09023 5 we?” 05053390 20.9 noon oounouuoum sum Eoum uoEonoquE odoc3 :9 Oman oEounoouxo .9mnns some :9 nous cwououm Hmsomouofia can noaonOuo«E 090:3 :2 Oman oeounoouao mo n02u9>2uos vaudoomn or» soon ooumHommuuxo na3 omen oEOusoouho mo ooflumuucoocoo one .ooosuoz can mamwuounm.uoocs omnwuoooo no oOEuoHuom ouo3 masons nodumxwm ucoaoamaoo m 2H990990299IH92¢ 99H3.99209090H2 99093 2H om¢9 9209900990 90 m99H9099 20H9¢NH9 9292999200 .Hm magmas 82 QN fi|||1|sm .sm magmas 3.95 To... .83 92852.5 ON a. N. no u 1 u q d 888 00 900 ()3de .LNBINB'IOWOO 96 83 24'40 noEOnOHOfia Houucoo Eoum woodman?“ 9.33»qu omvm oEounoouho IIIIIV unsouou09e 021m 593 00.39.39 99939.39 mcvm oEounoouao 20-9 noEonouowa umm Eoum powmwusm aaasfiuumm omvm oEousooumo .wmnnm sumo Ga nous caououm 9mEOnouoHE can omvm oEOHnoouxo wwwwwusa adamfiuumm mo nO9u9>9uoo owmaoomn Eouu oocfisuouoo was Oman oEOucOOuao mo coflumuucoocoo one .noonuoz can mandamus: woos: confluonmo no omfiuomuom ouo3 masons scauoxflm newsmamsoo m 2H990990299IH92¢ 99H3 omcm 9209900990 99H9H999 999¢H99¢9 90 999H9099 20H9¢NH9 9292999200 .NN magmas 84 .- magmas .22... a.0... down. mzomzoots 0.9 0.0 06. 0.9 0.N 0.. a a a q q a 1a fl 1 1 \I\ 4 ‘ av \\\\\ o \‘ 4 \\ 4 \‘fi 4 o ‘\\\h-‘ .llllll.\.\\ 4 6 L o m 9 N (13de .LNBWB-IdWOO % C) 00 I—u—l C) Q 85 antibody bound by cytochrome P450 from PB microsomes and that by cytochrome P450 from control microsomes. This might be eXplained if the spectrophotometric assay of cytochrome P450 is not valid. It has recently been pro- posed that apoprotein exists which cannot be detected by the spectrophotometric assay(51). If antibody binds to apoprotein which is not detected as cytochrome P450, then the amount of cytochrome P450 is underestimated and will give the results shown in Figure 21. It seems reasonable, therefore, that the spectrophotometric assay of cytochrome P450 should be scrutinized. If the percent of complement fixed is plotted against the concentration of cytochrome P450 partially purified from microsomes isolated from control and PB— and 3—MC- pretreated rats, the relationships observed in Figure 17 on the basis of microsomal protein are also Observed in Figure 22. Unfortunately the ratios of specific activity for cytochrome P450 from partially purified PB, 3-MC, and control microsomes do not correspond to the ratios Observed on the basis of amount of bound antibody (Table II). Again, the amount of cytochrome P450 can be underestimated if the assay is dependent upon heme bound to apoprotein. Evidence for the location of the enzymatic site with respect to antigenic sites has also been presented. The 86 results of agglutination and enzyme inhibition assays indicate that some antigenic sites for cytochrome P450 are exposed on the exterior surface of the membrane, i.e., on the cytoplasmic side. The agglutination process relies upon externally exposed antigenic sites. The failure to inhibit aminopyrine—N—demethylation suggests that the enzymatic site is buried within the membrane and is exposed upon solubilization. Preliminary studies by Frederick O'Neal in Dr. Aust's laboratory'with benzphetamine and benzpyrene hydroxylations in a reconstituted system show specific inhibition in the presence of anti—hemoprotein 3. Benzphetamine hydroxylation, specific for cytochrome P450 in PB-induced systems, was inhibited by the antibody but benzpyrene hydroxylation (specific for cytochrome P448) was not. The results imply that the antibody is specific for the 44,000 dalton cytochrome P450 and that antigenic sites exposed in a soluble, reconstituted system do inhibit enzy- matic reactions. In summary, the results presented in this thesis pro- vide further evidence that: l) anti-hemoprotein 3 is specific for the 44,000 dalton hemoprotein from microsomes induced in PB—pretreated rats and 2) the active site is not freely exposed to the exterior but probably buried within the membrane. If specific antibodies for each type 87 of cytochrome or hemoprotein can be made, it will be pos- sible to conduct studies specific for one component in the presence of other similar proteins. With the use of antibodies, one will be able to investigate the relative rate of turnover and enzyme specificity and, perhaps, develop more reliable assays for individual hemoproteins. REFERENCES 10. 11. 12. 13. 14. REFERENCES Ernster, L., and Orrenius, S. (1965) Fed. Proc., 33 1190. Kuntzman, R. (1969) An. Rev. Pharmacol., 2, 21. Conney, A.H. (1967) Pharmacol. Rev., 12, 317. Gillette, J.R. (1971) Metabolism,‘gg, 215. Brodie, B.B., Gillette, J.R., and LaDue, B.N. (1958) An. Rev. Biochem.,‘gz, 427. Brown, R.R., Miller, J.A., and Miller, E.C. (1954) J. Biol. Chem., 209, 211. ‘Williams, Jr., G.R., and Kamin, H. (1962) J. Biol. Chem., 237, 587. 5. Klingenberg, M. (1958) Arch. Biochem. Biophys., 376. Garfinkel, D. (1958) Arch. Biochem. Biophys., 11, 493. Omura, T., and Sato, R. (1964) J. Biol. Chem., 239, 2370. Omura, T., and Sato, R. (1964) J. Biol. Chem., 239, 2379. Gillette, J.R., Davis, D.C., and Sasame, H.A. (1972) An. Rev. Pharmacol.,.13, 57. 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Vol. XI, (Hirs, C.H;W., ed.), p. 928, Academic Press, New Ybrk, N.Y. 44. 45. 46. 47. 48. 49. 50. 51. 91 Rabat, E.A., and Mayer, M.M. (1961) Experimental Immunochemistry, Ch. 4, Thomas, Springfield, Illinois. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (1971) Biochemistry,.1g, 2606. Lowry, 0.3., Rosebrough, N.J., Farr, A.L., and Ran- dall, R.J. (1951) J. Biol. Chem., 193, 265. Imai, Y., and Sato, R. (1967) Europ. J. Biochem.,.1, 419. Goodfriend, T.L., Levine, L., and Fasman, G.D. (1964) Science, 144, 1344. Bashore, R.A., Facog, B.J., and Cdblence, C. (1970) Obs. and Gyn., 36, 391. Hook, G.E.R., Bend, J.R., Heel, D., Fouts, J.R., and Gram, T.E. (1972) J. Phar. Exp. Ther., 182, 474. Siekevitz, P. (1973) J. Supramol. Struc.,.l, 471. APP’IX APPENDIX In Review: Ann F. Welton, Fredrick 0. O'Neal, Linda C. Chaney and Steven D. Aust, "Multiplicity of Cytochrome P450 HemOproteins in Rat Liver Microsomes: Preparation and specificity of an antibody to the hemoprotein induced by phenobarbital," J. Biol. Chem. Abstracts: A.F. Welton, L.C. Mandorf, and S.D. Aust, "Studies of Multiple Cytochrome P450 Hemoproteins in Rat Liver Microsomes," Fed. Proc.,‘gg, 1437 (1974). R.W. Moore, F.O. O'Neal, L.C. Chaney, and S.D. Aust, "Specificity of Antibody to the Cytochrome P450 Hemoprotein Induced by Phenobarbital,“ Fed. Proc. (in press). 92