63] Isolation of plasma membranes from adipocytes

Abstract

This chapter describes the isolation of plasma membranes from adipocytes. There are several commonly used methods for the isolation of adipocyte plasma membranes. On the basis of marker enzyme activities, it is estimated that these plasma membrane preparations contain 20%–30% endoplasmic reticulum contamination. It is found that an adipocyte plasma membrane preparation containing less than 9% endoplasmic reticulum contamination can be obtained. This increase in purity is of particular value for experiments aimed at investigating physiological changes in the composition and molecular organization of the plasma membrane of adipocytes. Collagenase preparations causing complete dispersion of fat depots into intact cells that respond to 10 −5 M epinephrine with an appropriate increase in lipolysis rate are used. An aliquot of fat cell suspension is used to obtain purified plasma membranes. By measuring the trichloroacetic acid-precipitable 125 I in these plasma membranes and the protein content, the counts per minute of [ 125 I]DDISA per milligram of plasma membrane protein can be calculated.