Isolation of ribosome containing synaptosome subpopulation with active in vitro protein synthesis

Abstract

Subpopulations of synaptosomes harvested from neonatal rat brain cortices revealed a differential ability to synthesize protein in vitro. Incubation of synaptosomes with radiolabeled leucine, followed by continuous sucrose gradient centrifugation produced an asymmetric shift in the radioactivity toward the higher density sucrose fractions. The bulk of the mitochondrial cytochrome c-oxidase activity was also found in these fractions, however, subfractionation studies of osmotically-lysed synaptosomes suggested that the newly-synthesized proteins reside in an osmotically sensitive, non-mitochondrial compartment. The ability of each subpopulation of synaptosomes to synthesize protein in vitro was assessed after their isolation from linear continuous sucrose gradients. There was an enrichment of highly active protein synthesizing particles in the "heavy" subpopulations of neonatal synaptosomes. The inhibitory effects of chloramphenicol and cycloheximide on the protein synthesis in these particles were similar to those of the original synaptosome fraction. Electron microscopic analysis revealed an increase in the numbers of ribosome-containing structures resembling dendritic and axonal growth cones.