Isolation of Plasma Membrane Vesicles from Fat Cells of Epididymal Fat Pads of the Rat by Aqueous Two-Phase Partition

Abstract

The responses of fat cells to insulin involve potentially important membrane alterations. There is now considerable evidence to support the concept that insulin stimulates glucose transport by inducing a rapid and reversible translocation of glucose transporters from an intracellular membrane pool to the plasma membrane [1–4]. However, little is known about the cellular mechanism of insulin and the process or biochemical events whereby glucose transporter levels at the cell surface may be modulated. Initial efforts [5] to better characterise the glucose transport system involved use of a sensitive silver staining method to visualise membrane proteins in two-dimensional gel electrophoretograms in conjunction with computerised scanning and quantification. A 90 kDa protein was lost from the plasma membranes upon incubation of fat cells with insulin and calcium. We are investigating the dynamics and identity of the 90 kDa protein in the fat cell, and especially the mechanism whereby the protein is dissociated from the plasma membrane as well as its fate and function in relation to the oncoming glucose transporter vesicles from the cytoplasm.