Journal of Lipid Research, Vol 18, 32-36, Copyright © 1977 by Lipid Research, Inc.
Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes
VC Joshi, AC Wilson and SJ Wakil
The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC
1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC
1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a
simple method for routine assay of the terminal enzyme based on
complementation of the enzyme with chick embryo liver microsomes lacking
desaturase activity. Desaturation of [1-14C]stearoyl CoA by the
enzyme-microsome mixture is then assayed by thin-layer chromatography of
the reaction products and determination of the amount of oleate formed.
Microsomes from the livers of starved-refed rats were used as the source of
the stearoyl CoA desaturase. The enzyme alone, solubilized and free from
cytocrome b5 reductase and cytochrome b5, was unable to catalyze the
desaturation of stearoyl CoA. However, after preincubation with chick
embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was
active. The enzyme activity was linear with time and desaturase protein
under the conditions described and depended on the concentrations of Triton
X-100 present in the preincubation and the assay. The optimum
concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15%
in the assay. The desaturation activity was dependent on NADH and O2, and
was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in
this method eliminates the need to use purified cytochrome b5 reductase,
cytochrome b5, and liposomes for routine assays and greatly reduces the
complexities of timing and order of addition encountered in the existing
assays.
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