Abstract
A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.
Highlights
A procedure for the preparationof monospecific an- satility of the liver microsomal enzymesystem anddifferences tibodydirectedagainst rat livermicrosomalcytoin metabolism dependingon the age, sex, strain, and treatment chrome P-450. is described.Thisantibody,together of an animal
The antibody titer to the contaminant was very low relative to the titer against cytochromeP-450
The lack of monospecificity of anti-P-450. was only apparent when double immunodiffusion analysis was performed under highly sensitive conditionsas described under “Experimental
Summary
Cytochrome P-450, from Aroclor 1254-treated rats is highly purified (2, 18), anti-P-450, recognizes a t least one other microsomal protein. It isknown that antibodies can be produced to contaminating protein present at no more than. A cross-reaction of antibodies with heterolopeptide is identical inmolecular weight with theprotein gous antigens could result in inhibition of catalytic activity stripped by KSCN from a control IgG Sepharose immunoaf- withoutimmunoprecipitation.Cytochromes P-450,, P-450b, finity column and is identified as the heavy chain of IgG by and P-450, are efficient catalysts of the 7a-hydroxylation of its mobility. Regulation of Cytochrome P-450 and Epoxide Hydrolase presence of anti-P-4501,and anti-P-450, These results show the marked specificity of the three monospecific antibodies for their respective antigens. Unknown cytochrome P-450 is derived from the arithmetic difference between 100'%,and the sumof cytochromes P-454, 1'-4501,,and P-450
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