Highly Purified Synaptosomal Membranes from Rat Brain: PREPARATION AND CHARACTERIZATION

Abstract

Synaptosomal plasma membranes were isolated from whole rat brain by a method derived from several existing ones. The procedure involves the sequential centrifugation of a crude mitochondrial fraction through a discontinuous sucrose gradient and a continuous cesium chloride gradient. The material is then subjected to osmotic lysis and centrifuged through an additional discontinuous sucrose gradient. The synaptosomal membranes prepared in this way contain only very small amounts of mitochondria, endoplasmic reticulum, myelin, axonal and glial plasma membranes, and lysosomes; the level of contamination was estimated by assay for appropriate marker enzymes, and by assaying the final membrane preparation for radioactivity following the addition of previously labeled radioactive contaminants at the start of the isolation procedure. On the basis of these criteria, the contaminants examined appear to account for less than 10 to 15% of the protein in the preparation. The isolated membranes were sequentially extracted with Triton X-100 and sodium dodecyl sulfate; as previously described, the Triton-soluble fraction contains those portions of the synaptosomal membrane not associated with the synaptic junction, whereas the sodium dodecyl sulfate dissolves the junctional complex. The fractions were subjected to electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, and the gels were stained for protein with Coomassie brilliant blue. The results indicate that some proteins are common to both fractions, but that each fraction contains several unique protein species. The membrane preparation also contains RNA and DNA in limited amounts. The bulk of the RNA resembles mitochondrial RNA with respect to its mobility in polyacrylamideagarose composite gels.