58] Purification of ribosomes from Euglena gracilis

Abstract

Publisher Summary This chapter discusses Euglena gracilis and its two distinct classes of ribosomal particles, one found in the cytoplasm and the other in the chloroplasts. Euglena cultures grown in the dark result in cells free of chloroplasts and chloroplast ribosomes. Exposure of the dark-grown (colorless) cells to light induces the synthesis of the proteins of the photosynthetic apparatus and the plastid structure, accompanied by the appearance of the chloroplast ribosomes. Cytoplasmic ribosomes are isolated from green, colorless, and permanently bleached cells of E. gracilis. This chapter describes isolation of cytoplasmic ribosomes and chloroplast ribosomes. Ribosomes of a higher degree of purity are obtained by a deoxycholate treatment. A critical step in the procedure for the isolation of chloroplast ribosomes is the deoxycholate treatment of the chloroplasts. The cytoplasmic ribosomes can be distinguished from the chloroplast ribosomes by four criteria: (1) sedimentation characteristics of the particles; (2) base composition and sedimentation characteristics of the ribosomal RNA; (3) biochemical activity as demonstrated by the in vitro incorporation of amino acids into protein; and (4) distribution in the chloroplast structure. Chloroplast ribosomes cannot be demonstrated in dark-grown or permanently bleached cells.