The biosynthesis of cytochrome P‐450 in vitro

The biosynthesis of cytochrome P450 by rough endoplasmic reticulum in vitro

“…The rats used in these experiments were kept and treated with phenobarbital as in [7]. Rough endoplasmic reticulum was prepared from the livers of these animals [13].…”

“…Cytochrome P450 was purified from the livers of phenobarbital-treated rats [8] and used to raise antibodies in New Zealand white rabbits [7,8]. The antibodies obtained are specific for cytochrome P450 as judged by Ouchterlony analysis, ability to precipitate spectrally-detectable cytochrome P450 from solubilised membrane and electrophoretic analysis of immunoprecipitates [7,8].…”

“…The antibodies obtained are specific for cytochrome P450 as judged by Ouchterlony analysis, ability to precipitate spectrally-detectable cytochrome P450 from solubilised membrane and electrophoretic analysis of immunoprecipitates [7,8]. Radiolabelled polypeptides immunoprecipitated from subreticular fractions after protein synthesisin vitro have the same electrophoretic mobility as the cytochrome P450 used as antigen to prepare the antibodies [8 1.…”

“…Protein synthesis in vitro was carried out as in [7] but using "r(Xabelled ammo acid hydrolysate (56 mCi/matom) 4 &i/ml. Incubations were supplemented with a non-radioactive mixture of those amino acids not present in the hydrolysate.…”

“…However, we have recently demonstrated that a part of the newly-biosynthesised cytochrome P4.50, a major membrane protein, can be found inside vesicles derived from the rough endoplasmic reticulum after protein synthesis in vitro [7,8]. Newly-biosynthesised cytochrome P450 has also been located inside vesciles derived from rough and smooth endoplasmic reticulum after incorporation of radiolabelled amino acids in situ by perfusion of the liver [8].…”