Faculty Publications

Comparative Immunochemical Studies of Cytochrome P‐450CAM of Pseudomonas putida and of Cytochrome P‐450SCC, of Bovine Adrenocortical Mitochondria

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European Journal of Biochemistry





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Antibodies raised in rabbits against highly purified preparations of two cytochromes (cytochrome P‐450CAM from Pseudomonas putida and cytochrome P‐450scc from bovine adrenocortical mitochondria) were found to be specific for their respective antigens in Ouchterlony doublediffusion assays. In each case a single sharp curve was observed and no response was seen with other P‐450 hemeproteins. In enzyme inhibition studies with reconstituted monooxygenase systems both the precipitating antibodies and soluble, monovalent Fab−1 fragments obtained from them by peptic digestion were effective inhibitors. In contrast to the Ouchterlony assay, anti(P‐450CAM) not only stopped camphor hydroxylation completely but also caused measurable inhibition of the cholesterol side‐chain cleavage (desmolase) reaction while anti(P‐450scc) inhibited camphor hydrolxylation significantly in addition to abolishing desmolase activity. Iodination of cytochromes P‐450CAM and P‐450scc with the help of chloramine‐T produced soluble, radiolabeled preparations with specific activities ranging from 3 × 108 to 1 × 109 counts × min−1×μmol−1 which showed undiminished enzymatic activities and gave a single precipitin curve each in the Ouchterlony assay with anti(P‐450CAM) and anti(P‐450scc), respectively. In the iodinated preparations 60–70% of the tyrosine residues were found to be present as monoiodotyrosines while no other amino acid was labeled. A sensitive radioimmunoassay was developed, measuring inhibition of binding between 125I‐labeled cytochrome P‐450CAM and its antibodies by cross‐reacting material, which revealed substantial cross‐reactivity of cytochromes P‐45011β (88%), P‐450scc (64%) and P‐450LM‐2 (60%). Conversely, competitive binding o.125I‐labeled cytochrome P‐450sccand unlabeled cross‐reacting antigens to anti(P‐450scc demonstrated cross‐reactivities for cytochromes P‐450sccβ (60%), P‐450CAM (75%) and P‐450LM2 (90%). Incubation of cytochrome P‐450scc with adrenodoxin, and of cytochrome P‐450CAM with putidaredoxin, prior to interaction with anti(P‐450scc) and anti(P‐450CAM), respectively, caused marked decreases in the ability of the corresponding P‐450 hemeproteins to bind to their antibodies. Even the BrCN‐derived hemepeptides of cytochromes P‐450CAM and P‐450ssc were still able to recognize the antibodies elicited by their parent proteins as evidenced by significant crossreactivities in the radioimmunoassay. It is concluded that the four P‐450 hemeproteins tested in this investigation, cytochromes P‐450CAM, P‐450ssc, P‐45011β, and P‐450LM‐2, have one or more antigenic determinants in common and that a major antibody binding site is associated with the hemepeptide. Copyright © 1980, Wiley Blackwell. All rights reserved

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