Purification of aminoacyl-tRNA synthetase kinase activities associated with threonyl- and tyrosyl-tRNA synthetases isolated from Bom:NMRI mouse liver.
Aminoacyl-tRNA synthetase kinase activities and aminoacyl-tRNA synthetase activities prepared from postribosomal supernatants of Bom:NMRI mouse liver comigrated through the following purification steps: 1 a) gel filtration on Sephadex G-200, 1 b) chromatofocusing, 2) affinity chromatography on immobilised total tRNA (mixture of tRNA's, specific for all aminoacyl-tRNA synthetases) and 3) affinity chromatography on immobilised tRNA, specific for each of threonyl- and tyrosyl-tRNA synthetases. The purification factors for threonyl- and tyrosyl-tRNA synthetases were about 17000x. The purified synthetases showed only one protein band following sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified threonyl- and tyrosyl-tRNA synthetase proteins were found also to possess threonyl- and tyrosyl-tRNA synthetase kinase activities, respectively. The purification of tRNA(Thr) and tRNA(Tyr) as well as a method for the renaturation and identification of threonyl- and tyrosyl-tRNA synthetase activities in protein bands obtained by SDS PAGE are described.
Department of Biochemistry, University of Bergen, Norway.
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Berg
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013914:Threonine-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism
D011494:Protein Kinases; C081588:threonyl tRNA synthetase kinase; C081589:tyrosyl tRNA synthetase kinase; D014448:Tyrosine-tRNA Ligase; D013914:Threonine-tRNA Ligase