Neonatal imprinting and hepatic cytochrome P‐450 II. Partial purification of a sex‐dependent and neonatally imprinted form(s) of cytochrome P‐450

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Abstract
A new form of cytochrome P‐450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P‐450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P‐450 is either absent or present in low concentrations in cytochrome P‐450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N‐demethylase activities of this partially purified cytochrome P‐450 revealed that the presence of testosterone 16α‐hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N‐demethylase, testosterone 7α‐and 6β‐hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P‐450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α‐ and 7α‐hydroxylase activities in a concentration‐dependent manner, without impairing 6β‐hydroxylase activity. These data suggest that the partially purified cytochrome P‐450 from adult male rats consists of both imprintable (16α‐) and nonimprintable (7α‐) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P‐450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P‐450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P‐450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α‐and 7α‐hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P‐450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P‐450 accounts for a small fraction of the bulk of total cytochrome P‐450. However, the existence of this form of cytochrome P‐450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.