Sedimentation behaviour of aminoacyl-tRNA synthetases from mixed lysates of yeast and rabbit liver.
The subcellular distribution of five aminoacyl-tRNA synthetases from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated. To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles. The vacuole-free supernatant was subjected to sucrose density gradient centrifugation. No evidence for multimolecular associations of these enzymes was found. In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction. A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit. The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes. The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis. Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013045:Species Specificity; D013104:Spheroplasts / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology
D000604:Amino Acyl-tRNA Synthetases