Synthesis of cytoskeletal proteins in bulk-isolated neuronal perikarya.

Abstract

Abstract: Neuronal perikarya were isolated from young rat brain by sucrose density gradient centrifugation of the tissue, dissociated with a low concentration of trypsin. The isolated cells retained their endogenous proteins, and were capable of active protein synthesis. After incubation with L‐[35S]methionine, perikarya were homogenised and separated into soluble and particulate fractions by centrifugation at 70,000 g. Newly synthesised polypeptides in each fraction were resolved by SDS‐gel and two‐dimensional gel electrophoresis coupled with fluorography. Neuronal perikarya synthesised predominantly actin, and α1‐, α2 and β‐tubulin. In addition, polypeptides with molecular weights of 35,000, 68,000 and 85,000 were heavily labelled. On two‐dimensional electrophoresis, microheterogeneities were seen in soluble actin as well as in soluble tubulins, indicating that heterogeneities reported for brain actin and tubulins are inherent in neuronal actin and tubulins, but not owing to the heterogeneity of cells in the brain tissue. Structural differences between soluble tubulins and those associated with the particulate fraction were indicated by two‐dimensional gel electrophoresis and also by one‐dimensional peptide maps. The 68,000 molecular weight polypeptide synthesised in neuronal perikarya in vitro yielded a peptide map virtually identical with that generated from the major component of the neurofilament triplet polypeptides that were synthesised in situ. The 160,000 and 200,000 components of the neurofilament triplet were also synthesised in perikarya in vitro, but to disproportionately weaker extents compared with the 68,000 component.