JBC, Vol. 254, Issue 2, 408-413, Jan, 1979
Mixed function oxidases in sterol metabolism. Separate routes for electron transfer from NADH and NADPH
R. D. Crowder and D. R. Brady
Oxidative deformylation of
4-hydroxy[14C]methylene-5alpha-cholest-7-en-3-one and oxidative
demethylation of [30,31-14C]4,4-dimethyl-5alpha-cholest-7-en-3beta-ol by
rat liver microsomes have been compared with regard to the manner in which
electrons are introduced from both NADH and NADPH. Evidence suggests that
NADH and NADPH support oxidation of both substrates via separate routes of
electron transfer. Thus, 10 micron cytochrome c will inhibit
NADPH-supported oxidation to 40 to 50% of control activity leaving
NADH-supported oxidation unaffected. Also, treatment of microsomes with
subtilisin diminishes NADPH-supported oxidation to 10 to 30% of control
activity for either substrate to 70 to 90% of control activity while
NADH-supported oxidative activity is virtually unaffected. Studies on the
oxidase activities and NADPH-cytochrome c reductase as well as
NADH-ferricyanide reductase have shown marked differences in activity in
the presence of inhibitors. Thus, 9 mM 2'-AMP inhibits NADPH-cytochrome c
reductase to 10 to 20% of control activity while NADPH-supported oxidative
demethyl ation and deformylation are essentially unchanged. Mersalyl at 15
to 25 nmol/mg of microsomal protein inhibits both reductases to 20 to 40%
of control activity; oxidative demethylation is unaffected and oxidative
deformylation stimulated slightly when NADPH is used. Finally, antibody to
NADPH-cytochrome c reductase inhibits oxidase activity for either substrate
to 70 to 90% of control activity while reductase activity is inhibited to
10 to 30% of control activity.
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