Improved preparative methods for isolation and purification of tobacco chloroplast ribosomes, ribosomal proteins, and rRNA

Abstract

Publisher Summary This chapter describes the procedures for the isolation of chloroplast ribosomes on a preparative scale by zonal sucrose gradient centrifugation from the whole leaf extracts. Methods for extracting ribosomal proteins and ribosomal RNA (rRNA) from the purified ribosomes are also described. The protein-synthesizing machinery of chloroplasts is a class of ribosomes that are unique to the chloroplast and of a 70 S type with many structural and functional similarities to bacterial ribosomes. The prokaryotic-like ribosomes are present in green leaves in equimolar amounts, a relative to the 80 S cytoplasmic ribosomes that share common features with other eukaryotic cytosolic ribosomes. Chloroplast ribosomes differ from the 80 S cytosolic ribosomes in their susceptibility to dissociation into subunits at different levels of magnesium ion. Chloroplast ribosome monosomes can be isolated at higher magnesium concentrations. The magnesium concentration required to control the equilibrium between the monosome and its dissociated subunits depends on the concentration of the major monovalent cation present in the solution.