An investigation of the antigenic determinants on chloroperoxidase and purified rat liver microsomal cytochrome P-450b.
Chloroperoxidase (CPO) exhibits many physicochemical and catalytic properties similar to those of the bacterial and microsomal cytochromes P-450. Therefore, the possible similarities between the antigenic determinants of CPO and rat liver microsomal cytochrome P-450b were investigated. Polyclonal antibodies against CPO and rat liver cytochrome P-450b were raised in rabbits and used to investigate the antigenic cross-reactivity between CPO and P-450b. Although anti-CPO antibodies were capable of inhibiting the ethyl hydroperoxide-supported N,N-dimethylaniline (DMA) demethylation activity of CPO by more than 80%, they were unable to inhibit the NADPH-supported demethylation of DMA by cytochrome P-450b in the reconstituted system. The ethyl hydroperoxide-supported demethylation of DMA by CPO was not affected by the addition of anti-P-450b antibodies which inhibited cytochrome P-450 activity greater than 90%. In order to probe for the possible existence of common antigenic determinants which were not involved in catalytic activity, the cross-reactivities were investigated using enzyme-linked immunosorbent assays. There was no cross-reactivity between anti-CPO and cytochrome P-450b, or anti-P-450b and CPO using enzyme-linked immunosorbent assays. When control, phenobarbital-, isosafrole-, and beta-naphthoflavone-induced rat and rabbit liver microsomes and CPO were analyzed by Western blotting and developed with anti-P-450 antibodies, only the phenobarbital- and isosafrole-induced microsomes showed a positive reaction in the P-450 region. When anti-CPO antibodies were used on Western blots of the same series of proteins, a positive reaction was observed only with CPO.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002711:Chloride Peroxidase / Q000276:immunology*; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology*; D000939:Epitopes / Q000032:analysis*; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010544:Peroxidases / Q000276:immunology*; D011817:Rabbits; D051381:Rats
D000939:Epitopes; D007106:Immune Sera; D007074:Immunoglobulin G; D003577:Cytochrome P-450 Enzyme System; D010544:Peroxidases; D002711:Chloride Peroxidase