Effects of thiol concentration in the assay of liver microsomal acylCoA cholesterol acyl transferase.

AcylCoA cholesterol acyl transferase of the liver is usually evaluated by incubation of the microsomal fraction with a labelled long chain acylCoA, mainly oleoylCoA. A number of authors carry out the assay in the presence of dithiotreitol, despite the fact that a lower activity was reported in the presence of the thiol. Data reported here show that increasing dithiotreitol concentration in the assay medium results in a progressive decrease of the enzyme activity. Correspondingly, an increase of the substrate hydrolysis occurs up to 5 mM dithiotreitol. At 10 mM concentration of the thiol, complete inhibition of both the esterification by ACAT and the hydrolysis occur. Radioactivity associated with polar compounds increases with dithiotreitol up to the highest tested concentration (10 mM). With heat inactivated microsomes no formation of these compounds is observed.