Topographical studies of chick brain synaptic plasma membrane polypeptides

Abstract

The topographical distribution of polypeptides of chick synaptic plasma membrane was studied using pyridoxal phosphate and sodium borotritide labeling, lactoperoxidase-catalysed iodination, and neuraminidase and galactose oxidase-sodium borotritide labeling. Two-dimensional polyacrylamide gel electrophoresis of the labeled polypeptides gave additional data regarding the arrangement of the polypeptides in the synaptic membrane. Pyridoxal phosphate labeling of intact synaptosomes was confined to the external surface, without labeling the synaptoplasmic polypeptides, only by careful adjustment of the reaction conditions. Fourteen major polypeptides were labeled. These had apparent molecular weights of 210,000, 160,000, 130,000. 100,000, 92,000, 82,000, 60,000, 52,000, 42,000. 34,000, 29,000, 26,000, 24,000 and 19,000 daltons. These proteins had previously been shown to be labeled by neuraminidase and galactose oxidase-sodium borotritide labeling of intact synaptosomes. Therefore these results confirmed previous data. The results also showed that the probe, pyridoxal phosphate, did not label any polypeptides other than those labeled by neuraminidase and galactose oxidase-sodium borotritide. Radioactivity patterns of pyridoxal phosphate labeling in one-dimensional polyacrylamide gels showed a majority of the radioactivity incorporated into three peaks of apparent molecular weights 42,000, 29,000 and 26,000 daltons. Autoradiographs of two-dimensional polyacrylamide gels showed these proteins to be basic, indicating that they may have a great number of lysine residues relative to acidic residues. Lactoperoxidase-catalysed iodination of intact synaptosomes and subsequent two-dimensional polyacrylamide gel electrophoresis of the membrane polypeptides showed most of the radioactive label to be incorporated into a streak corresponding to an apparent molecular weight of 55,000 to 62,000 daltons, partly overlapping the alpha-tubulin subunit. There is a good correlation between the labeling patterns of one-dimensional gels and two-dimensional gels, with the exception of high molecular weight proteins of molecular weights 195,000, 185,000, 150,000, 140,000 and 130,000 daltons which are clearly separated only in the two-dimensional labeling pattern. Galactose oxidase-sodium borotritide labeling showed almost all the external polypeptides to be glycoproteins. Neuraminidase treatment of synaptosomes before galactose oxidase incubation increased the level of 3H incorporation several-fold, indicating the presence of sialic acid groups of many glycoproteins. Neuraminidase and galactose oxidase- I sodium borotritide labeling of isolated membranes labels a glycoprotein of apparent molecular weight 73,000 daltons which is not labeled in the intact synaptosomes, suggesting that a major portion of this protein is exposed to the internal surface of the synaptic membrane. Galactose oxidase-sodium borotritide labeling of synaptoplasmic polypeptides is not increased by neuraminidase pretreatment, indicating that sialic acid groups are not present in the synaptoplasmic polypeptides.