Extraction of proteins from the large subunit of bovine mitochondrial ribosomes under nondenaturing conditions.
The 55 S mammalian mitochondrial ribosome (referred to hereafter as "mitoribosome") is protein-rich, containing nearly twice as much protein as the Escherichia coli ribosome. In order to produce soluble mitochondrial proteins and protein-deficient subribosomal particles for use in functional and structural studies, the proteins of bovine mitoribosomes were extracted by washing in a series of buffers containing increasing concentrations of LiCl as the only chaotropic agent. LiCl disruption is used in order to preserve the solubilized proteins in a substantially "native" configuration. The extraction mixtures were characterized by sucrose density gradient analysis and the compositions of the stripped protein and residual pellet fractions were determined by two-dimensional polyacrylamide gel electrophoresis. In order to analyze the behavior or individual proteins, the intensity of Coomassie blue stain for each protein was normalized against the intensity of stain for the same protein in a control sample. Buffers with 1, 2, and 4 M LiCl each extract a specific subset of mitoribosomal proteins, while another group of proteins remains in the residual pellet fraction. Although very few proteins are detected in only one condition, most proteins are specifically enriched in one fraction. This LiCl procedure, therefore, produces fractionated groups of mitoribosomal proteins which can be used directly as a source for those proteins in which they are enriched, or they can be used as a starting point in further purification procedures. In contrast to results with E. coli ribosomes, several mitoribosomal proteins remain core-associated, indicating a different structural organization in these ribosomes.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002712:Chlorides; D004591:Electrophoresis, Polyacrylamide Gel; D008094:Lithium; D018021:Lithium Chloride; D046911:Macromolecular Substances; D008722:Methods; D008928:Mitochondria / Q000032:analysis*; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000032:analysis*
D002712:Chlorides; D046911:Macromolecular Substances; D012269:Ribosomal Proteins; D008094:Lithium; D018021:Lithium Chloride