Abstract
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 125I-labeled P-450 PB-4 and also in vitro-synthesized 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 one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-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 and other P-450 isoenzymes present in rat liver endoplasmic reticulum.
Highlights
From the Department of Cell Biologyand Kaplan Cancer Center, New York University Medical Center, New York, New York 10016and the §Department of Biological Chemistry and Dana-Farber Cancer Institute, Harvard Medical School, Boston,Massachusetts 02115
The total number of P-450 isoenzymes expressed in a given tissue is synthesized apo-P-450 PB-4t,he yields of immunopre- not known but atleast 10 individual forms have to date been cipitation were low relative to thoabt tained using anti- isolated from rat liver
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 PB4
Summary
HydrophilicityAnalysis of P-450 PB-4-A hydropathy profile (Kyte andDoolittle, 1982) was calculated for P-450 PB-4 (using the amino acid sequence of Fujii-Kuriyama et al (1982) as modified byYuan et al (1983))to help identify hydrophobic and hydrophilic segments suitable for the preparation of sitespecific antipeptide antibodies (Fig. 1).The extreme aminocolumns as described under “Materials and Methods” (see Fig. S-6). Lys Lys Ser G1u Ala Phe Met Pro Phe Ser Thr Gly Lyr A. rg-Ile Cys Leu Gly Glu Gly 440. Asp I l e Ala LYS I l e 480 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 thfeact that itis directed against asegment of P-450 PB-4which is proline-rich (Pro2*Pro%; 40% proline). IgG (e.g. 15% precipitation efficiency with affinity-purified antipeptide antibody 10 versus 36% precipitation efficiency using total anti-P-450 PB-4 totalIgG). In order to ascertain whether the antipeptide antibodies couldrecognize apocytochrome P-450 PB-4, a wheat germ extract was programed with mRNA derived from phenobarbital-induced rat liver andthe affinity-purified IgGswere used to immunoprecipitate in vitro synthesized P-450 PB-4. 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
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