Journal of Lipid Research, Vol 23, 345-351, Copyright © 1982 by Lipid Research, Inc.
Partial purification and characterization of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and 5 beta-cholestane-3 alpha, 7 alpha-diol 27- monooxygenase
Y Atsuta and K Okuda
Hepatic mitochondrial cytochrome P-450 has been partially purified from the
non-induced rat liver. The purification consisted of solubilization with
cholate, polyethylene glycol fractionation, chromatographic separation
using omega-amino-n-octyl Sepharose 4B column, and chromatography on
hydroxylapatite. The overall purification of the enzyme from the
solubilized extract was about 22-fold on the basis of specific content of
cytochrome P-450, and 50-fold on the basis of specific activity. The
partially purified enzyme was active for both 5 beta-cholestane-3 alpha, 7
alpha, 12 alpha-triol and 5 beta-cholestane- 3 alpha, 7 alpha-diol. That
the enzyme activities for these substrates were not due to two different
enzymes but to the same active site of a single enzyme protein was shown by
several pieces of evidence, i.e., behavior to thermal inactivation,
pH-dependency of the reaction velocities, experiments with mixed
substrates, and behavior towards inhibitors and activators. The lower Km
value and the higher Vmax for 5 beta-cholestane-3 alpha, 7 alpha, 12
alpha-triol compared to 5 beta- cholestane-3 alpha, 7 alpha-diol seem to be
important factors for the regulation mechanism that keeps the ratio of
cholic acid/chenodeoxycholic acid constant in rat bile.
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