Isolation of Plastid Ribosomes from Euglena


The chapter discusses the isolation of plasmid ribosomes from Euglena. The chloroplastic ribosomes of Euglena are unstable; if exposed to suboptimal ionic conditions, the 68 S monosome is converted to a 53 S particle. Under the ionic conditions used to stabilize the chloroplast monosome, organelles isolated in 12 mM Mg2+ clump together, making it impossible to free them of cytoplasmic contamination. Chloroplast ribosomes cannot be isolated directly from whole-cell lysates, therefore, a procedure is developed for the large-scale isolation of structurally intact chloroplasts. If the structural integrity of the chloroplast is maintained, the chloroplast ribosomes are protected from the unfavorable ionic conditions of the chloroplast isolation buffers; these chloroplasts are thus suitable starting material for the isolation of chloroplast monosomes. The purity of each chloroplastic ribosome preparation is checked on sucrose gradients or on polyacrylamide gel after extractions of the rRNAs because the quantity of contaminating cytoplasmic ribosomes is variable. Chloroplastic monosomes are separated in pure form by centrifugation on sucrose gradients in tubes or in zonal rotors. Chloroplast ribosomal subunits are obtained by dialyzing the chloroplast ribosomal pellet against low Mg 2+ (1.0 mM) before separation on sucrose gradients. © 1979, Elsevier Inc. All right reserved.

Publication Title

Methods in Enzymology