Altered leucyl-transfer RNA synthetase from a mammalian cell culture mutant.
Altered leucyl-tRNA synthetase from a mammalian cell culture temperature-sensitive mutant, tsHl, was compared with enzyme from normal wild type Chinese hamster ovary cells. The mutant enzyme had a Km for leucine four times larger than that of wild type and enzyme levels 3-10% that of wild type. The presence of tRNA was necessary during in vitro heating of the mutant enzyme to allow expression of thermolability while the presence of tRNA protected wild type enzyme against thermal inactivation. The tsHl enzyme was stable when heated alone or in the presence of tRNA, leucine, and ATP simultaneously. The mutant's enzymes aminoacylated tRNALeu, tRNAVal, and tRNAIle with fidelity in vitro as determined by cochromatography of the amino-acyl-tRNA isoacceptors on RPC-5 reversed phase chromatography. The mutant failed to show any defect other than the direct formation of leucyl tRNALeu by leucyl-tRNA synthetase.
D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002460:Cell Line; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine; D007700:Kinetics; D007930:Leucine; D007935:Leucine-tRNA Ligase / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D011188:Potassium / Q000494:pharmacology; D012343:RNA, Transfer; D013696:Temperature; D014633:Valine
D007532:Isoleucine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase; D007930:Leucine; D014633:Valine; D008274:Magnesium; D011188:Potassium