The Structure of Phenobarbital-inducible Rat Liver Cytochrome P-450 Isoenzyme PB-4 PRODUCTION AND CHARACTERIZATION OF SITE-SPECIFIC ANTIBODIES*

Fifteen peptides corresponding in sequence to seg- ments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the an- tipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demon- strated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 1251-labeled P-450 PB-4 and also in uitro-synthesized apo-P-450 PB-4, the yields of immunopre- cipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibod- ies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5.

The Structure of Phenobarbital-inducible Rat Liver Cytochrome P-450 Isoenzyme PB-4 PRODUCTION AND CHARACTERIZATION OF SITE-SPECIFIC ANTIBODIES* (Received for publication, June 17, 1985) Alan B. FreyS, David J. Waxman$, and Gert Kreibichll From the Department of Cell Biology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016 and the §Department of Biological Chemistry and Harvard Medical School,Boston,Massachusetts 021 15 Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 1251-labeled P-450 PB-4 and also in uitrosynthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal onethird of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed 0-deethylation of the model substrate 7ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 j3NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 *This work was supported in part by Grant BC-462 from the American Cancer Society (D. J. W.) and National Institutes of Health Grants GM 21971 (G. K.) and GM 20277 (D. Sabatini). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
li Recipient of a Career Development Award from the Irma T. Hirschl Charitable Trust. To whom correspondence and reprint requests should be addressed. and other P-450 isoenzymes present in rat liver endoplasmic reticulum.
Cytochrome P-450 (P-4501), the hemoprotein component of the hepatic mixed function oxidase system, catalyzes the oxidative metabolism of a wide variety of lipophilic endogenous substrates and xenobiotics. These metabolic reactions yield water-soluble products which are readily eliminated by urinary or biliary secretion (Gillette et al., 1972). This capacity for metabolism of a broad spectrum of substrates is due to the existence of several isozymic forms of P-450, each of which exhibits broad but unique substrate specificity profiles (Guengerich, 1979;Lu and West, 1980;Conney, 1982). The total number of P-450 isoenzymes expressed in a given tissue is not known but at least 10 individual forms have to date been isolated from rat liver (reviewed by Waxman, 1985). These isoenzymes can be grouped into gene families, each containing several related P-450 isoenzymes (e$ Adesnik and Atchison, 1985). Thus P-450 PNF-B and P-450 ISF-G, the two major rat hepatic P-450s induced by P-naphthoflavone and isosafrole, respectively (Reik et al., 1982;Guengerich et al., 1982;Goldstein et aL, 1982) are members of one gene family while P-450s PB-4 and PB-5, the two major isoenzymes induced by treatment of rats with phenobarbital (Waxman and Walsh, 1982;Ryan et al., 1982b), belong to another gene family (Atchison and . Detailed structural studies have shown that these latter two P-450s are closely related and, in fact, differ from each other by only 14 (out of 491) amino acids (Fujii-Kuriyama et al., 1982;Yuan et al., 1983). Analysis of a cloned rat genomic library suggests that there are at least seven other cross-hybridizing genes in this family (Atchison and  and recent studies of the rabbit P-450 system have identified several cDNAs which may be members of yet a third P-450 gene family (Leighton et al., 1984).
It has been postulated that a relatively invariant "framework" or series of domains characterizes the members of a particular P-450 gene family with sequence variation largely restricted to "variable" segments of the polypeptide chain of P-450 PB-4 was analyzed by the method of Kyte and Doolittle (1982) using a scan length of 9 amino acids. Shown are the segments calculated to be hydrophobic ( p e a k above the mid-line) and hydrophilic (troughs). The locations of the peptides selected for synthesis are indicated by bars at the top of the graph and the numbers next to the bars designate the peptide number. (Kumar et al., 1983). These variable segments are likely to contribute to the substrate specificities and reactivities characteristic of each P-450 form and thus may correspond to those domains of the polypeptide involved in substrate binding. On the other hand, the heme binding site and the sites of interaction of P-450 with NADPH cytochrome P-450 reductase and cytochrome b, may correspond to more highly conserved segments of the polypeptide chain. Although the anchoring of P-450 to the endoplasmic reticulum is probably mediated by hydrophobic segments of the polypeptide chain, these segments need not necessarily be conserved between different P-450 forms. Identification of the postulated variable segments within members of a P-450 family should help establish the structural basis for the unique specificity profiles exhibited by closely related isoenzymes. Conversely, identification of segments of sequence homology between more distantly related P-450s may help elucidate the importance of common functional domains for P-450 structure and catalytic capacity. This article describes a study designed to investigate the structure of rat liver P-450 PB-4 and immunochemically related isoenzymes. The chemical synthesis of 15 peptides of P-450 PB-4 and the use of these peptides for production and characterization of site-specific antibodies is described. These antipeptide antibodies are shown to be effective probes useful for identification of segments important for catalytic activity as well as for delineation of regions of functional and structural homology common to the 'multiple P-450 isoenzymes found in rat hepatic tissue.

MATERIALS AND METHODS AND RESULTS~
Hydrophilicity Analysis of P-450 PB-4-A hydropathy profile (Kyte and Doolittle, 1982) was calculated for P-450 PB-4 (using the amino acid sequence of Fujii-Kuriyama et al. (1982) as modified by Yuan et al. (1983)) to help identify hydrophobic and hydrophilic segments suitable for the preparation of sitespecific antipeptide antibodies (Fig. 1). The extreme amino-terminal region of P-450 PB-4 was thus found to be highly hydrophobic in character (average hydropathy (h) = 2.96 for residues 3-20) consistent with its proposed function in directing the nascent apoprotein to the endoplasmic reticulum (Bar-Nun et al., 1980). This amino-terminal segment is more hydrophobic than most cleavable NHz-terminal signal sequences contained in pre-secretory proteins (Waxman and Walsh, 1982), suggesting that it may help anchor the P-450 to the endoplasmic reticulum. Although as many as eight additional hydrophobic segments are suggested by these analyses ( Fig. S-1; also see Heineman and Ozols, 1982;Tarr et al., 1983) none exhibits the high hydrophobicity characteristic of transmembrane segments in general or of P-450 PB-4 residues 3-20 in particular.
Peptide Synthesis and Antibody Production-Peptides representing both hydrophobic segments of P-450 PB-4 (peptides 1, 6, 8, 10, 12, and 14) and more hydrophilic segments (peptides, 2, 3, 4, 5 , 7, 9, 11, 13, and 15) were synthesized using a benzhydrylamine resin and the method of Merrifield (1963) and then purified as describedunder "Materials and Methods" (Fig. 2, Fig. S-2, and Table I). The synthetic peptides varied from 6 to 31 residues and exhibited average hydropathy values ranging from highly hydrophobic ( h = 2.2; peptide 8) to highly hydrophilic ( h = -2.2; peptide 9). These peptides, numbered sequentially from the amino to the carboxyl terminus, correspond in sequence both to P-450 PB-4 and to the highly homologous P-450 PB-5. One exception, however, is peptide 15 which contains the Thr at position 407 characteristic of Peptides were conjugated to carrier proteins in a carbodiimide-catalyzed reaction (see "Materials and Methods") and then used for immunization of rabbits and mice. Since some of the peptides were insoluble in aqueous buffers all of the conjugation reactions were performed in mixtures of organic solvents consisting of dimethylformamide/CHzC12/NaHC03, No underivatized carrier protein was detectable by amino acid analysis (data not shown) and by SDS-PAGE ( Fig. S-3), suggesting quantitative coupling of the peptides under the conditions employed. A mixture of derivatized peptide and nonconjugated peptide adsorbed onto activated charcoal was used as immunogen.
Antibody production was monitored by ELISA as shown in Figs. S-4 and S-5. Antipeptide antisera were tested for reactivity against the immunizing antigen as well as against heterologous peptides and purified P-450 PB-4. Each antipeptide antibody was found to react specifically with the corresponding peptide antigen as well as with purified P-450 PB-4. One notable exception was antipeptide antibody 1 which reacted both with peptide 1 and with the peptide partially overlapping in sequence, peptide 2 ( Fig. S-4). Antipeptide IgGs were affinity purified from sera on peptide-Sepharose columns as described under "Materials and Methods" (see Fig. S-6). As shown in Table I there was a general correlation between serum titer and recovery of affinity-purified IgG. No correlation was apparent between the predicted conformation or average hydropathy of a given peptide and the titer of the corresponding antisera.
Reactivity of Antipeptide Antibodies with P-450 PB-4-Each of the affinity-purified antipeptide antibodies was assayed for its reactivity with both native and denatured P-450 PB-4. The affinity-purified antibodies all recognized the purified isoenzyme but exhibited somewhat different reactivities as determined by ELISA assay (Fig. S-7). The antipeptide antibodies were also tested for their ability to recognize SDSdenatured P-450s PB-4 and PB-5 by Western blot analysis (Fig. 3). Only antibody 2 reacted well with both microsomal and purified P-450 PB-4 (and P-450 PB-5) on the Western blots; the other antipeptide antibodies did not exhibit comparable reactivities even at low sera dilutions (1:25). The good reactivity of antipeptide 2 in the Western blot analysis may reflect both its high serum titer and the fact that it is directed against a segment of P-450 PB-4 which is proline-rich (Pro2*-Pro%; 40% proline). The effectiveness of the antipeptide antibodies at immunoprecipitation of purified P-450 PB-4 was examined in the presence of non-ionic detergent. lZ5I-labeled P-450 PB-4 was incubated with each affinity-purified antipeptide antibody in the presence of Nonidet P-40 and the immune complexes then recovered with Protein A-Sepharose. Samples were then analyzed by SDS-PAGE and the fraction of immunoprecipitable P-450 PB-4 determined by y-counting after excision of the radiolabeled P-450 PB-4 bands. All of the antipeptide antibodies tested3 were active at immunoprecipitation of purified P-450 PB-4 ( Fig. 4) with negligible immunoprecipitation observed when using either preimmune sera or sera which had been pretreated with the cognate peptide. The efficiency of immunoprecipitation by the antipeptide antibodies was, however, much lower than that obtained with anti-P-450 PB-4 Antibody 14 was generally inactive; see e.g. Table I. IgG (e.g. 15% precipitation efficiency with affinity-purified antipeptide antibody 10 versus 36% precipitation efficiency using total anti-P-450 PB-4 total IgG).
In order to ascertain whether the antipeptide antibodies could recognize apocytochrome P-450 PB-4, a wheat germ extract was programed with mRNA derived from phenobarbital-induced rat liver and the affinity-purified IgGs were then used to immunoprecipitate in vitro synthesized P-450 PB-4. An example of the results is shown in Fig. S-8 where it is seen that the PB-4/PB-5 apoprotein is recognized specifically by the antipeptide antibodies. Once again anti-P-450 PB-4 heterosera were significantly more efficient than the antipeptide antibodies at immunoprecipitation of apocytochrome P-450 PB-4 (data not shown).

Reaction of Different Anti-P-450 PB-4 Heterosera with the Synthetic
Peptides-Rabbit antisera were raised both against P-450 fraction C (isolated as described by West et al. (1979) and corresponding to P-450 PB-4 containing a 20% PB-5 contaminant) (antibodies Ia-Id in Table 11) and also against PB-4 which was isolated by subjecting fraction C to preparative SDS-PAGE as the final step of the purification (antibodies IIa, IIb). Individual synthetic peptides were used as antigens in an ELISA assay to probe the nature of the antigenic determinants recognized by each of these anti-P-450 PB-4  Sequence data taken from Fujii-Kuriyama et al. (1982). In peptide 1 the Thr 4 at position is corrected to Ser * As predicted by Chou and Fasman (1978). None indicates that no prediction concerning the structure can be Serum titer measured at exsanguination and tested by ELISA using peptides conjugated to a carrier protein Affinity purification of the antipeptide antibodies was performed as described under "Materials and Methods."

made.
different than that used for immunization (see "Materials and Methods").
The data are represented as micrograms of affinity purified ("AP") IgG recovered per 20 ml of serum.
heterosera. Four of the peptides were found to react with the 6 heterosera examined ( Table 11). The epitopes represented by these 4 peptides were not major antigenic determinants of P-450 PB-4 since the reactivities of the heterosera towards the peptides could be completely removed from each of the sera by adsorption with the appropriate peptide-Sepharose matrices without significantly decreasing the antisera titer assayed using purified P-450 PB-4 (data not shown).
Recognition of Other P-450 Isoenzymes by Antipeptide Antibodies-The antigenic relatedness of P-450 PB-4 and other P-450 forms was assessed by the cross-reactivity of the antipeptide IgGs with other P-450 isoenzymes in an ELISA assay ( Table 111). Each of the antipeptide antibodies was found to be equally reactive with P-450 PB-4 as with P-450 PB-5. This result is, however, not surprising in view of the high amino acid homology between these two major phenobarbital-induced isoenzymes (Fujii-Kuriyama et al., 1982;Waxman and Walsh, 1982;Yuan et al., 1983). Significant cross-reactivity was also observed between several of the antipeptide antibodies and P-450 PNF-B, the major P-450 isoenzyme present in polycyclic-induced rat liver. Since these analyses were performed the amino acid sequence for P-450 PNF-B has been published (Kawajiri et al., 1984). A comparison of the amino acid sequences for the regions corresponding to the four most highly cross-reactive segments (peptides 2, 4, 9, and 12 of P-450 PB-4; Table 111) is shown in Table 1V. Considerable homology between these two isoenzymes is apparent for the segment corresponding to peptide 4. Thus the tetrapeptide Tyr-Gly-Asp-Val is conserved between the two isoenzymes as is the overall distribution of charged and nonpolar residues. Similarly, the sequences Pro-Pro-Gly-Pro and Leu-Pro (residues 31-34 and 37-38, respectively, peptide 2) and residues 295-296, 298-299, and 302 in peptide 12 are shared between these two P-450s (Table IV). By contrast, the highly charged segment corresponding to peptide 9 has more limited sequence homology to the corresponding segment of the PNF-B isoenzyme (residues 187, 189, and 192). The cross-reactivity between the two isoenzymes may reflect a similarity of local conformation in these regions. This supposition is supported by comparison of the hydropathy profiles for the two isozymes which locate the position of the corresponding segment in P-450 PNF-B at regions of similar hydrophilicity. Additional homologous segments corresponding to the synthetic peptides were not apparent upon inspection of the amino acid sequence of cytochrome P-450 PNF-B.
Inhibition of Monooxygenase Activity of Purified PB-4 by Antipeptide Antibodies-Each antipeptide antibody was evaluated for its ability to inhibit the in vitro metabolism of 7ethoxycoumarin catalyzed by purified P-450 PB-4 in a reconstituted system ( Fig. 5 and Table V). Each of the antipeptide antibodies (or control IgG) was incubated with purified P-450 PB-4 following which the hemoprotein was reconstituted with P-450 reductase, cytochrome b5, and lipid. These studies demonstrated that antibodies to three of the peptides (4, 6, and 7) were highly inhibitory in this system while antibodies 2,9, 10, 11, and 13 were partially inhibitory. The remaining antibodies exhibited either no effect on catalytic activity (5, 8, and 15) or were somewhat stimulatory (12 and 14).

DISCUSSION
Chemically synthesized peptides have been employed for the production of antibodies useful for the study of exon usage (Nathans and Hogness, 1983), production of vaccines to viruses (Shinnick et aL, 1982), investigation of functionally important regions of enzymes or receptors (Evin et al., 1984;Van Eldik et al., 1983;Komoriya et al., 1984) as well as for studying the nature of the immune response (Niman et al., 1983). Antibodies to synthetic peptide immunogens often, but not always (Bittle et al., 1982), react with the corresponding intact protein, thereby permitting valuable information to be learned concerning the structure and function of the parent protein (Walter and Doolittle, 1983). The use of a well-defined peptide immunogen has the virtue of avoiding the immunodominance of select epitopes often seen when immunization is conducted using whole proteins Walter and Doolittle, 1983). Western blot analysis of liver microsomes prepared from untreated, phenobarbital-induced or 0-naphthoflavone-induced adult male rats (8 pg each, lunes 1-3, respectively) as well as purified P-450s PB-1, PB-4, and PB-5 (0.2 pg each, lunes 4-6, respectively) performed as described under "Materials and Methods." Antiserum to peptide 2 was diluted 200-fold into 10 mM KPi, pH 7.4, 0.9% (w/v) NaCl containing 10 mg/ml bovine serum albumin and then incubated 3 h a t 22 "C with the nitrocellulose blot. Shown is a photograph of the blot following immunoperoxidase staining using 4-chloro-1-naphthol as substrate. Subsequent repeat probing of the same blot with rabbit anti-P-450 PB-1 heterosera confirmed the presence of immunoreactive PB-1 in lunes 1-4.
In this paper, we have described the preparation and characterization of site-specific antibodies using peptide antigens corresponding in amino acid sequence to the nucleotide sequence of the cloned cDNA of rat liver cytochrome P-450 PB-4. The synthetic peptides included in this study correspond to both hydrophilic and hydrophobic portions of P-450 PB-4 and together comprise almost 40% of the entire protein sequence. The synthetic peptides were examined for their reactivity with anti-PB-4 antisera (raised against either the apoor holoenzyme) in order to probe the antigenic structure of the intact P-450. Four of the peptides (peptides 2, 9, 15, and a peptide comprised of residues 408-421; peptide 16) were thus found to be included in antigenic sequences presented by the parent protein (Table 11). Since antipeptide activity IO I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 Antibody rwnber

FIG. 4. Reactivity of antipeptide IgGs towards '2SI-labeled P-450 PB-4.
Iz5I-labeled P-450 PB-4 was dissolved in a buffer containing 50 mM Tris-HC1, pH 7.4, 190 mM NaC1, 6 mM EDTA, 2.5% Triton X-100, 0.02% NaN3, and 100 units/ml Trasylol and immunoprecipitated with affinity-purified antipeptide IgG (0.035 mg). Following recovery of immune complexes with Protein A-Sepharose (0.05 ml of a 50% suspension), the immunoprecipitates were analyzed by SDS-gel electrophoresis (8% polyacrylamide) followed by radioautography. The radioactivity in the P-450 PB-4 bands was then determined as described under "Materials and Methods." The insert shows an autoradiograph obtained upon immunoprecipitation using antipeptide 2 IgG (lune a). Lune b shows immunoprecipitates recovered with IgG which had been preincubated with 0.1 mg of peptide 2 (60 min, 37 "C) and lane c shows an immunoprecipitate recovered using preimmune IgG. 0.035 mg of rabbit IgG was used to derive the immunoprecipitate shown marked as C.
could be completely adsorbed from these heterosera with only a small decrease in titer, these peptides probably represent minor antigenic determinants. Alternatively, these four peptides may represent small segments of major antigenic determinants in which case their strengths as epitopes in the intact protein would be difficult to assess.
The synthetic peptides were also used to raise antipeptide antisera in rabbits. Since each peptide was used to immunize a single animal no firm conclusions concerning the relative immunogenicity of each peptide can be made. Each antibody was characterized by several criteria as to its ability to react with the peptide immunogen and also with the parent P-450. Each of the antipeptide antibodies reacted with P-450 PB-4 as determined by ELISA assay and also as assessed by immunoprecipitation of lZ5I-labeled P-450 PB-4. Although the immunoprecipitation reaction was shown to be specific, the precipitation yields using affinity purified antipeptide IgG were typically only 515%. These values can be compared to a 35% precipitation efficiency using anti-P-450 PB-4 total IgG. Low yields were also obtained following immunoprecipitation of in vitro synthesized apo-P-450 PB-4. Low efficiencies of immunoprecipitation by antipeptide antibodies have also been reported by others (cf. Mariotini et al., 1983), suggesting that antibodies produced against a single epitope

TABLE I1
Reactivity of different preparations of anti-P-450 PB-4 IgG towards synthetic peptides Heterosera raised against P-450 PB-4 were tested for their reactivity with the synthetic peptides by ELISA as described under "Materials and Methods." Data are expressed as the minimum amount of IgG (in micrograms) which gave a reaction greater than twice background (assayed against bovine serum albumin as antigen) where + = 10 pg of IgG, ++ = 2.5 pg of IgG, +++ = 1.2 pg of IgG, and ++++ = 0.6 pg of IgG. Blanks indicate no reactivity detected at 10 pg of IgG. * Antibodies Ia-Id are IgG preparations obtained from antisera of 4 rabbits, each immunized with the same preparations of P-450 fraction C (West et al., 1979) consisting of a mixture of about 80% P-450 PB-4 and 20% P-450 PB-5. e Antibodies IIa and IIb were obtained by immunizing two rabbits with P-450 PB-4 which was isolated from P-450 fraction C by SDS-PAGE.

TABLE I11
Isozymic cross-reactivity of antipeptide antibodies The reactivity of each of the affinitypurified antipeptide antibodies with purified P-450 PB-4 was compared to its reactivity with P-450s PB-5 and PNF-B in an ELISA assay. Purified P-450s were bound to microtiter plates and the ELISA assay performed as described previously (Waxman, 1984). Shown are the relative reactivities (expressed as a percentage) for each antipeptide antibody with PB-4 relative to PB-5 or PNF-B. Results are expressed as average f S.D.
for three independent experiments. Typical A415 values for PB-4 (determined after 9-fold dilution into 0.5% SDS) ranged from 0.4 to 0.9 A in these experiments using 2.5 pg of each affinity purified antibody/analysis. Control values (0.05-0.10 A ) corresponding to no antigen or no first antibody were subtracted from each sample. The isozymic cross-reactivity of an anti-P-450 PB-4 rabbit heteroserum is shown for comparison at the bottom. All the antibodies tested exhibited significant cross-reactivity with P-450 PB-5. Significant cross-reactivities to P-450 PNF-B are marked by asterisks (*). A415 values for antipeptides 8,13, and 14 were too low in these experiments to Dermit a meaningful determination of their cross-reactivity. may be less likely to participate in immune complex formation for reasons related to the availability of a limited number of antigen binding sites. Alternatively, the binding of an antipeptide antibody to its corresponding parent protein might be inherently of lower affinity than that of antiprotein heterosera due to differences in the manner of peptide-antigen presentation to the host. It has been proposed that the binding of antibodies to discontinuous antigenic determinants of proteins contributes significantly to immunoprecipitation by heterosera (Walter and Doolittle, 1983). The absence of such nonsequential determinants may partly account for the low efficiency of P-450 precipitation by the antipeptide antibodies. Finally, if, as suggested (Sutcliff et al., 1983;Niman et al., 1984), short peptides adopt various stable conformations in solution, the antipeptide antibodies used in this study may be comprised of antibodies principally directed against conformations different from those found in the parent P-450 PB-4. Although most of the antipeptide antibodies reacted with P-450 PB-4 in an ELISA assay only one (antipeptide 2) gave a good signal with either purified or microsomal P-450 PB-4 in a Western blot analysis. Possible explanations for the low reactivity of the other antipeptide antibodies on the Western blots include: (a) low titer of many of the antipeptide antibodies as compared to anti-P-450 PB-4 heterosera; (b) lower affinity of antipeptide antibodies in generak4 and (e) low reactivity of the antipeptide antibodies to denatured P-450. Antipeptide antibodies were used to evaluate the possibility that other P-450 isoenzymes share antigenic determinants with P-450 PB-4. As expected, each of the antipeptide antibodies reacted equivalently with P-450 PB-4 and the highly homologous P-450 PB-5. Significant cross-reactivities between four of the antipeptide antibodies and P-450 PNF-B, the major aromatic hydrocarbon-inducible P-450 were also observed. Several of the antipeptide antibodies also react with other P-450 isoenzymes purified from uninduced rat liver, consistent with the recent demonstration using anti-P-450 heterosera that some of these isoenzymes have antigenic determinants in common (Waxman, 1984). These finding demonstrate the utility of site-specific antibodies in the analysis of P-450 isoenzymes such as PB-4 and PNF-B, which do not cross-react when analyzed using conventional immunochemical methods.
The antipeptide antibodies were used to investigate the involvement of specific amino acid sequences in substrate metabolism. Three antibodies were significantly inhibitory in this assay (antipeptides 4, 6, and 7; Fig. 5), suggesting that the corresponding amino acid sequences are important for some aspect of substrate metabolism. The absence of significant inhibition by the other 12 antipeptide antibodies is not likely to reflect the inaccessibility of their respective epitopes for IgG binding since, for example, antipeptide antibody 15 binds specifically both to purified P-450 PB-4 and to the surface of microsomes derived from phenobarbital-treated rats, yet does not inhibit substrate metabolism? Inhibition of catalytic activity by antipeptides 4, 6, and 7 was less pronounced when the antibodies were added after reconstitution of the P-450 with cytochrome P-450 reductase, cytochrome bs, and lipid (data not shown). This finding suggests that the corresponding amino acid segments might not be involved directly with substrate binding but, rather, might facilitate productive interactions between the P-450 and cytochrome bs and/or P-450 reductase.
Anti-P-450 antibodies have been used by a number of investigators to identify structurally related P-450 isoenzymes. Thus heterosera raised to P-450b (equivalent to P-450 PB-4) do not distinguish isoenzyme PB-4 from the closely related PB-5 (Waxman and Walsh, 1982;Ryan et al., 1982a).

FIG. 5. Inhibition of P-450 PB-4-mediated 7-ethoxycoumarin 0-deethylation by affinity-purified antipeptide IgG. 7-
Ethoxycoumarin metabolism was assayed in vitro as described under "Materials and Methods." The antibodies used in this experiment were affinity purified. Antibody A refers to antipeptide antibody 7A, raised to a peptide corresponding to residues 145-158 of P-450 PB-4. Antisera raised to P-45Oc (P-450 PNF-B) has been shown to include a subpopulation of antibodies equally reactive with P-450d (P-450 ISF-G) (Reik et al. 1982). These latter crossreactivities have recently been confirmed and extended using a series of monoclonal antibodies to P-45Oc (Thomas et al., 1984). Structural studies have demonstrated a high degree of homology between these two forms (Sogowa et al., 1984;Haniu et al., 1984). Recent studies in our laboratory have shown that even after adsorption and affinity purification, anti-P-450 PB-1 and anti-P-450 2c both retain substantial activity against P-450 2d, a female-specific P-450 isoenzyme (Waxman, 1984). Finally, monoclonal antibodies to rabbit liver P-450 form 1 have been used to investigate the spatial distribution of antigenic determinants in the native enzyme as well as the cross-reactivity of P-450 1 with other forms of rabbit liver P-450 (Reubi et al., 1984). Although use of monoclonal antibodies to study P-450 structure and function has the TABLE V Inhibition of 7-ethoxycoumarin 0-deethylation by antipeptide antibodies 7-Ethoxycoumarin 0-deethylation was assayed for purified and reconstituted P-450 PB-4 as described under "Materials and Methods." Shown are the activities (expressed as per cent activity relative to control IgG) obtained in a representative experiment in the presence versus absence of 50 fig of affinity purified antipeptide IgG. Catalytic activity for the uninhibited reaction was 2.9 nmol of 7hydroxycoumarin/min/nmol of P-450. Values higher than 100% (peptides 3, 14, 12) indicate stimulation of catalytic activity. advantage of uniform affinity for all the antibody molecules, it is difficult to identify the specific epitopes to which the monoclonal antibodies are directed since the antigenic sites may be comprised of discontinuous amino acids . It is likely that none of the monoclonal antibodies raised against P-450 PNF-B by Thomas et al. (1984) were directed against the tetrapeptides Tyr-Gly-Asp-Val (residues 62-65 of P-450 PB-4) or Pro-Pro-Gly-Pro (residues 31-34 of P-450 PB-4) since no cross-reaction with other phenobarbitalinduced P-450s was detected by these investigators. Recent studies on the molecular biology of cytochrome P-450 indicate that this enzyme may actually be comprised of a "supergene" family of related isoenzymes (Adesnik and Atchison, 1985) with the precise number of molecular forms of Site-specific Phenobarbital P-450 Antibodies P-450 (even within a single organism) difficult to estimate due to the presence of genetic polymorphisms (e.g. Rampersaud and Walz, 1983). Sequence "hypervariabilities" localized within relatively invariant framework regions may characterize P-450 gene families (Atchison and  and may contribute to the subtle differences in substrate specificity or affinity of individual P-450s. The demonstration of localized segments of high amino acid sequence homology between antigenically and functionally distinct P-450 isoenzymes suggests that such homologous segments may be important for conserved structural and catalytic functions, including the interaction of P-450 with cytochrome P-450 reductase, structure of the heme binding site, and overall topological disposition of P-450 within the phospholipid bilayer of the endoplasmic reticulum. We are currently utilizing the antipeptide antibodies described in this article to study in greater detail the structural and functional relatedness between different P-450 forms as well as the precise membrane topology of P-450 PB-4.  When t o t a l microsomes were prepared the procedure was simplified such that the initial post-mitochondrial supernatent was layered over a 2 M sucrose cushion. Following centrifugation as before the total microsomes were isolated from the top of the 2 M cushion and then applied to the Sepharose 28 column. P-450 Enzyme Purification and Nomenclature P-450 PB-1 , P-450 PB-4, P-450 PB-5, cytochrome b5, NADPH cytochrome P-450 reductase (Waxman and Walsh, 1982) and P-450 BNF-B (Guengerich and Martin, 1980) were purified to apparent homogeneity and detergent removed as described in the references indicated. contained 29.2% acrylamide and 0.8% bisacrylamide. Gels were polymerized using 0.025% tetramethylethylene-diamine (TEMED) and ammonium persulfate. Electrode buffer stock solution (diluted 1:5 for use) contained 0.25 M TRW0.385 M glycine (pH 8.4) and 0.1% SDS added from a 20% stock. Samples were prepared by boiling for 2 m i n i n SDS sample buffer. Gels were stained for 3 h i n 0.2% Coomassie Blue i n 50"6 methanol/7% acetic acid, and destained by diffusion overnight in 30% methanol/lO% acetic acid. method of Lowry e t a l . (1951) using bovine serum albumin as a standard.
Protein was measured by a modification (Markwell e t a l . 1978) of the Immuneprecipitation of Labelled Proteins 1978) except t h a t 1% NP40 was substituted for the SOS/Triton X-I00 detergent Immuneprecipitation was performed as described (Goldman and Blobel, mixture. Immuneprecipitates were analyzed by SOS-PAGE followed by autoradiography using a Cronex intensifying screen (DuPont Photo Products).

Quantitation of Immuneprecipitated Radioactivity Following SDS-PAGE
on the SOS gels after aligning the exposed X-ray film with the dried gel.
Radiolabelled bands containing jmuneprecipitated proteins were located The appropriate bands were excised and counted d i r e c t l y i n a gamma counter.
Electro horetic Transfer of Proteins from SOS Polyacrylamide Gels t o Nitroceflulose Fllters 1981) using peroxidase-conjugated goat anti-rabbit IgG a s second antibody and Western blotting was performed essentially as described (Burnette, 4-chloro-1-naphthol (0.18 mg/ml dissolved i n 6% methanol) and 0.015% Hz02 as substrate.

Synthesis of Peptides
The following amino acid side-chain protecting moieties were used: toys1 f o r Arg; im-toluene ortho sulfonyl for Cleavage and amino acid side chain deprotection of each of the peptide mg/ml) using a Peninsula HF apparatus (Penninsula Laboratories). In O' C i n the presence of anisole (1.2 mg/ml resin) and methylethyl sulfide (1 addition, 5% pyridine was present during cleavage of peptides Containing glutamic acid to prevent anisolation. The resin was dried i n vacuo, washed with cold anhydrous diethyl ether and the peptides extracted w i t h alternate washes of neat TFA and water. The extracted peptides were then diluted with water and lyophilized. Some of the hydrophobic peptides precipitated upon dilution w i t h water b u t could be recovered as a suspension and then lyophilized. One peptide was recovered a s an oil (peptide 10). Peaks were collected, lyophilized twice from water, and analyzed by high and Schlesinger, 19791 and amino acid analysis. Peptide 1 was purified to performance 1 iquid chromatography (HPLC), manual Edman degradation (Boehnert homogeneity by HPLC using an Altex Cq column w i t h a TFA-CH3CN elution protocol. Phenylthiohydantoin amino acid derivatives obtained after Edman a sodium acetate-CH3CN gradient as described [Hunkapiller and Hood, 1975).
degradation were analyzed by HPLC on a Hewlett-Packard 10846 instrument using All samples f o r amino acid analysis were hydrolyzed i n 0.3 m l of 6N HC1:50% proprionic acid for 18 h a t 118°C ( F r e i l l e e t a l . 19821, dried, reconstituted in 0.2 N sodium,citrate (pH 2.2) and analysed by amino acid analysis u s i n g a Liquimat I11 system (Spackman e t a l . 1958). For amino acid analysis of synthetic peptides conjugated to Sepharose resin, the resins were washed extensively with water, dried i n vacuo and then hydrolyzed. The hydrolysate was diluted w i t h sodium a c e t x e b u f f e r i n the hydrolysis tube and centrifuged to remove discolored material which was generated by hydrolysis of the Sepharose.

Coupling of Synthetic Peptides
Affinity Purification of Rabbit Antibodies One ml of peptide-Sepharose (about 0.3 g dry weight) was pre-treated batchwise with a complete elution scheme which was a s follows. All procedures were conducted a t 4°C. The resin was first washed sequentially with 10 volumes each of: PBSlO.5 M NaCl/O.l% Triton X-100, PBS, PBSI0.5 1. 1 NaCl and finally PES. Elution of specific antibody w i t h 2 volumes of 0.05 M glycine-HC1 containin 0.15 M NaCl (pH 2.2) followed immediately by 2 volumes of 4 M NaSCN (pH 7.57. The resin was neutralized by extensive washing with PBSlO.5 1. 1 NaC1. For aFfinity purification, sera were thawed on ice, made up M 0.5 M NaCl, 0.01 M EDTA, 0.001% phenylmethylsulfoRy1 fluoride, 0.1% Triton X-100, centrifuged (20,000 rpm f o r 15 min in a Beckman Ti60 rotor), ana then mixed end-over-end for 12 h a t 4°C with one ml of the pre-treated resin.
The resin was then washed sequentially w i t h 10 volumes each of: PBSlO.14 Triton X-lOO/O.Ol M EDTA, 0.05 M NaHC03 (pH 8.5) containing 0.5 M NaCl, 0.05 M sodium acetate (pH 4.5) containing 0.5 M NaCl and finally PBS. The resins were washed extensively w i t h PBS until the A280 of the wash was less than 0.03 and specific antibodies eluted as described above. Antibodies were immediately neutralized w i t h 0.5 M sodium phosphate (pH 7.5), centrifuged (10,000 rpm for 10 min in a Sorval HB-4 rotor) and dialyzed exhaustively against PES.
Antibodies were concentrated by reverse dialysis against s t o r e d a t -20% sucrose, centrifuged a t 15,000 x g for 10 min, made 30% i n glycerol, and Several methods for conjugating synthetic peptides to carrier molecules for imunization and production of antibodies were examined including: carbodiimide-catalyzed conjugation to polyethylene glycol-6000, glutaraldehyde Coupling t o various carrier proteins, and carbodiimide conjugation to carrier proteins.
In all cases the method of coupling did not influence the production o f positive antisera. Carbodiimide conjugation of carrier protein i s small and the completeness of conjugation could readily be peptides t o malealated hen lysozyme was used i n t h i s study because the monitored on SDS gels since the derivatized carrier displayed a distinctly lower mobility man the parent lysozyme.
In addition, the amino acid composition of lysozyme is known, therefore amino acid analysis of conjugated carrier plus peptides could be used as a method of quantitating the coupling reaction.
Conjugation of peptides t o non-derivatized keyhole limpet of peptides to carrier proteins w i t h glutaraldehyde occasonally resulted i n hemocyanin was also used as a method of immunogen preparation. Crosslinking insoluble aggregates.
reactions (so that 1 conjugate could be used for immunization and the other Peptides were conjugated t o 2 different carrier proteins in separate conjugate for assaying the production of antisera) as follows. 100 mg of lysozyme was dissolved i n 20 ml of 0.1 M Na2B407 (pH 9.3) and derivatizea by the drop-wise addition of maleic anhydride (100-fold molar excess over amino groups) in dioxane over 2 h. The pH of the reaction was maintained a t 9.3 with NaOH and a f t e r 2 h the solution was dialyzed verses 10 ml.~ triethanolamine (pH 101 for 2 h, then dialyzed against water overnight and finally lyophilized. The malealated lysozyme and non-derivatized Keyhole Limpet Hemocyanin were conjugated with individual peptides as follows. Carriers were dissolved in 4 ml DMF:0.002 M NaHC03 pH 5.5 (70/30) a t 0.5 mg proteinlml and l-ethyl-3-l3-dimethylaminopropyl) carbodiimide IEDC) was added Peptide-carrier conjugates were analyzed by SDS gels and by amino acid analysis.
There was no unconjugated lysozyme remaining after the reactions as judged by electrophoresis of coupled peptides on 15% SDS-PAGE. Some peptide conjugates precipitated upon dialysis and these samples were dispersed by brief sonication before preparation of imunogens or use in ELISA assays. circulating immunoglobulins (Steinberg and Reinersten, 1978). 0.1-0.5 volumes adjuvant t o 1 volume antigen) was administered by intraperitoneal injection into 2 month Old female mice. 14 days later another injection was given and a third injection on day 28. Ascites fluid generally appeared about a week after the final immunization and was collected from the abdominal cavity with a 18 gauge needle connected to a syringe. The ascites fluid was centrifuged (l0,OOO rpm for 10 min i n a Sorvall HB-4 rotor) and the lipids removed w i t h a Cotton swab. A l l sera were aliquoted and stored at -2O' C i n the presence Of 0.05% NaN3. All experiments reported here used rabbit anti-peptide antibodies unless noted otherwise. Sepharose 48 was used as affinity matrix and conjugated w i t h purified P-450 PB-4 exactly as described ( (Waxman and Walsh 19821. The 0-deethylation reaction was stopped a f t e r 15 min by the additio; of 0.05 ml of ZN HCl and the product then extracted w i t h 0.9 ml of CHC13. Following centrifugation (2 min a t 1000 rpml 0.6 ml of the organic phase was back-extracted with 2 ml of 30 mM Na2B407 (pH 9 . 3 ) . 7-hydroxycoumarin formation was quantitated by fluorescence (excitation at 370nm and emission a t 455nm) i n comparison t o known standards using a Perkin Elmer WF4 instrument.
In assays which were performed i n the presence of anti-peptide antibodies, the antibodies (or heter0sera)'were added to the enzyme mixture either before or after reconstitution with NADPH P-450 reductase, cytochrome b5 and dilauroylphosphatidyl choline as indicated.
In cases were antibodies were added prior to reconstitution the IgGs were mixed with were added and the constituents then allowed to reconstitute. In cases where purified P-450 PB-4 for 30 min a t 22°C following which the other components 30 min a t 22°C prior to addition of substrate and NADPH. the antibodies were added a f t e r reconstitution the mixture was incubated f o r ReSUl t S Peptide cleavage and analysis: Following cleavage from the resin w i t h anhydrous HF, the synthetic peptides were gel-filtered and subjected to amino acid composition analysis as described i n 'Materials and Methods' (Figure S-2).
Since hydrolysis O f some peptides was incomplete i n 6N HCL, 50% proprionic acid in 6N HCL Was used ( F r i e l l e e t a l . , 1 9 8 3 ) ; t h i s was shown t o provide Complete and reproducible hydrolysis under standard conditions. These data demonstrated chains occured during HF cleavage. Analysis of the peptides by that the syntheses were complete and that minimal deterioration Of side amino-terminal Edman degradation and HPLC revealed that all Of the syntheses purified to homogeneity by HPLC.
(except peptide 1) yielded one major peptide product; peptide 1 was further lmmuneprecipitation of in vitro synthesized P-45O(pB) by anti-peptide IgG IgG NI a 1 a2 a3 "-W front * a b c d a n t i -p e p t i d e antibodies. A wheat germ e x t r a c t was programmed w i t h mRNR The following antibodies were used: row a, pre-imnune IgG from r a b b i t 1; row b, a n t i -p e p t i d e 1; row c, anti-peptide 2 ; row d. a n t i -p e p t i d e 3.