Protein asymmetry in chick synaptosomal plasma membrane.

The topographical arrangement of chick synaptosomal plasma membrane polypeptides was examined by lactoperoxidase-catalyzed iodination and trypsin digestion of proteins exposed at the exterior surface. The ““I-labeling profile on sucrose gradients of lysed, subfractionated synaptosomes generally coincided with the activity of the membrane marker (Na+-K+)-activated ATPase, which suggested that labeling was confined to the synaptic plasma membrane. Two types of experiments confirmed that lactoperoxidase had not crossed the membrane to label the interior of the synaptosome. First, trypsin digestion of labeled synaptosomes altered the Coomassie blue-staining pattern of membrane polypeptides but not the pattern of internal synaptoplasmic polypeptides. Second, under conditions of extensive membrane labeling, internal polypeptides remained unlabeled. Additional experiments showed that iodination was restricted to the external surface: when synaptic plasma membranes were isolated before lactoperoxidase-catalyzed iodination or incubation with trypsin, all membrane polypeptides were labeled or digested, respectively. However, relatively few polypeptides were labeled or digested when intact synaptosomes served as substrate. This supported the contention that iodination was restricted to the external surface, and, further, suggested that all polypeptides are exposed at one or both membrane surfaces. Eleven polypeptides were labeled to varying degrees by subjecting intact synaptosomes to lactoperoxidase-catalyzed iodination. Three peaks contained about two-thirds of the radioactivity. These polypeptides which are exposed at the external surface have molecular weights of 130,000, 100,000, 92,000, 60,000, 82,000, 42,000, 34,000. 29,000, 26,000, 24,000, and 19,000. Trypsin digested about half of the polypeptides exposed at the exterior surface, but only those with the highest molecular weights.