Rapid and efficient purification of plasma membrane from cultured cells: characterization of epidermal growth factor binding.
We have devised a rapid and simple protocol for the purification of the plasma membrane from several lines of transformed cultured cells. A431 or KB plasmalemma was purified in 90 min with a two-step centrifugation cycle after selectively inducing microsomal aggregation by the addition of calcium to homogenized cells. Relative specific activity analysis using membrane marker enzymes on the various fractions indicated that the isolated plasmalemma was purified 8-12-fold over the starting homogenate and contained a high density of epidermal growth factor (EGF) receptors. Transmission electron microscopy showed the final membrane suspension consisted of unilamellar vesicles with an average diameter of approximately 100 A. The purified membrane vesicles avidly bound to 125I-EGF and reached equilibrium within 30 min. Microfiltration assays indicated more than 90% of the total binding can be displaced by excess unlabeled ligand. Equilibrium binding analysis showed a single class of high-affinity 125I-EGF binding site, with Kd = 0.14 nM and Bmax = 0.1 pmol/mg of protein for purified KB membrane and Kd = 1.2 nM and Bmax = 5.26 pmol/mg of protein for purified A431 membrane. Gel electrophoresis of 125I-EGF cross-linked to membrane EGF receptors showed a distinct autoradiographic band at 170 kilodaltons, which could be displaced with excessive amounts of unlabeled EGF. Finally, EGF-dependent autophosphorylation of the EGF receptor was clearly demonstrated with the purified membrane preparation. Membrane vesicles purified in this manner can be stored in liquid nitrogen for several months without losing their biological activity.
D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D004798:Enzymes / Q000032:analysis; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D008854:Microscopy, Electron; D010766:Phosphorylation; D014461:Ultracentrifugation / Q000379:methods
D004798:Enzymes; D066246:ErbB Receptors