Isolation of chromaffin cell plasma membranes on polycationic beads

We have tried to define which proteins of chromaffin cell plasma membranes are facing the cytoplasm by surface labelling a selectively oriented membrane preparation. Viable chromaffin cells were isolated by collagenase treatment of bovine adrenals. Plasma membranes from these cells were isolated on polycationic beads by the method of Jacobson and Branton (Jacobson, B.S. and Branton, D. (1977) Science 195, 302--304). The purity and orientation of the membranes were defined by biochemical and morphological criteria. The membranes, with their external side apposed to the bead surface, were enriched about 10-fold with respect to a whole cell homogenate, and contained only small amounts of contaminating organelles. Surface specific iodination of membranes on beads with 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodogen), followed by polyacrylamide gel electrophoresis, allowed the identification of cytoplasmically exposed proteins. A different pattern was observed when intact cells were labelled prior to membrane isolation. The advantages and possible uses of this immobilized membrane preparation are discussed.