General Discussion

Abstract

I would like to comment on the studies concerning the participation of cytochrome b₅ in the hydroxylation reactions. As Drs. Mannering and Sasame have indicated, they feel that this cytochrome only participates in the NADH stimulation of the hydroxylation. Similarly I believe that there is some kinetic evidence supporting their immunological evidence that cytochrome b₅ is not involved in the hydroxylations when NADPH is the sole source of reducing equivalents. A couple of years ago we published a study in the Archives of Biochemistry and Biophysics (150:227, 1972) in which we demonstrated that the activation energy of ethylmorphine N-demethylase was significantly higher than the activation energy of the cytochrome P-450 reductase in the presence or absence of ethylmorphine. This suggested to us that there are two steps of nearly equal rate which control the rate of the overall demethylase reaction. We tried to determine whether the other step could be the reduction of cytochrome b₅, but it is so rapid that we could not measure it. Hence, there would always seem to be enough of the reduced cytochrome around so that at least its reduction could not be rate limiting and this could not be the cryptic second slow step we were searching for.