Isolation and characterization of free and membrane-bound polyribosomes from rabbit spinal cord

Abstract

A procedure is described for the preparation of free and membrane-bound polyribosomes from rabbit spinal cord. First, myelin and filaments are floated away from other subcellular components. The latter are then fractionated by differential centrifugation followed by sedimentation through a discontinuous sucrose gradient. The ribosomal fractions are characterized by their electron microscopic appearance, RNA/protein ratios and sedimentation profile in a linear sucrose gradient. Both membrane-bound and free polyribosomes are active in incorporating amino acids in a cell-free system, whether heterologous or homologous pH 5 enzyme fractions are employed. Autoradiographic analysis of translation products separated by electrophoresis on SDS-polyacrylamide gels shows that both ribosome fractions are active in the synthesis of a variety of proteins including polypeptides which comigrate with α- and β-tubulins and actin. The utility of these polyribosome preparations for the cell-free study of protein biosynthesis is indicated by the high molecular weight of many of the translation products, and by the similarity of the electrophoretic pattern of translation products from the cell-free systems to the pattern of radioactive protein synthesized by rabbit spinal cord during the hour prior to sacrifice.