Physicochemical characterization of ribosome-like (55-S) particles from rat liver mitochondria

Abstract The putative mitochondrial ribosomes of rat liver (55-S particles) have been purified by zonal sedimentation of crude particles in low-salt (25 mM K + , 5.0 mM Mg 2+ ) and high-salt (150 mM K + , 3.0 mM Mg 2+ ) sucrose gradients, or by the use of a modified procedure involving sedimentation through discontinuous and linear gradients containing Brij-58. Under all conditions tested, the 55-S component exhibits a buoyant density in CsCl of 1.45 g · cm −3 . Up to 90 % of the 55-S particles could be dissociated asymmetrically into subunits of 39 S and 29 S following brief incubation at 37 °C in high-salt buffer; the purified large subunits exhibit the same low buoyant density as the parent 55-S particles ( ϱ CsCl = 1.45 g · cm −3 ). In identical analytical conditions, the large and small subunits of cytosol ribosomes band at 1.614 g · cm −3 and 1.550 g · cm −3 , respectively, and Escherichia coli 70-S ribosomes have a buoyant density of 1.638 g · cm −3 . It is calculated on the basis of the buoyant density that the 55-S particles and their subunits contain 75 % protein and 25 % RNA; using values of 0.50 · 10 6 and 0.30 · 10 6 daltons for the two major components of mammalian mitochondrial RNA, it is estimated that the ribosome-like 55-S particles are about 3.20 · 10 6 daltons in weight and are composed of subunits having weights of 2.0 · 10 6 and 1.20 · 10 6 daltons, respectively. It is concluded that the 55-S particles are considerably smaller than the 80-S ribosomes of liver cytosol (4.60 · 10 6 daltons), but no smaller and perhaps slightly larger than the 70-S ribosomes of E. coli (2.60 · 10 6 –2.70 · 10 6 daltons). The 55-S particles are inactive when assayed in a polyphenylalanine-synthesizing system. However, (a) they carry labelled peptide chains following incubation of intact mitochondria in conditions of protein synthesis, and (b) the labelling of the 55-S component is not affected by cycloheximide but is severely inhibited by chloramphenicol.