Microsomal Cholesterol 7α -Hydroxylase Phosphatases

Abstract

When rat liver microsomes are incubated at 37° in TRIS buffer, there is a time-dependent loss of the activity of cholesterol 7α- hydroxylase (1). This loss of activity can be prevented if the incubation is carried out in phosphate buffer or if fluoride is added to the TRIS buffer system. These results are consistent with the presence of a protein phosphatase in the microsomal fraction. To determine that this activity was not due to other subcellular fractions, we prepared microsomes by our standard procedure and also by the calcium precipitation technique of Kamath et al. (2) as modified by Goodwin and Margolis (3). These preparations were tested for the presence of enzyme activities considered to be markers for other subcellular fractions. The results of these assays are shown in Table I. It can be seen that although the mitochondrial fraction is somewhat contaminated with microsomal enzymes, there is less mitochondrial or lysosomal contamination in our microsomal fraction.