Lysyl transfer ribonucleic acid synthetase (L-lysine:tRNA ligase (AMP), EC 6.1.1.6) was purified to a state of apparent homogeneity from bakers? yeast. For each of two different preparation procedures, a 250- to 500-fold purification of the enzyme was obtained, and the final purification step yielded two distinct protein components with specific activity values of approximately 0.5 and 1.0 micromole of lysyl-tRNA formed per min per mg of protein at 30?. The major component showed the higher specific activity in the aminoacylation reaction, was free from aminoacyl-tRNA synthetases other than lysyl-tRNA synthetase, gave a single protein boundary on sedimentation in the analytical ultracentrifuge, and showed one band on polyacrylamide gel electrophoresis. Both enzyme components appeared to be acidic proteins. Amino acid analysis of the major component showed a high content of aspartic and glutamic acid residues. The two protein components were distinguished by their difference in electrophoretic mobility on starch and polyacrylamide gels and by position of elution on ion exchange columns. Both components catalyzed lysine-dependent exchange of 32P-pyrophosphate into ATP in the absence of lysine-acceptor RNA, each showing the same specific activity value of approximately 3 micromoles of inorganic pyrophosphate exchanged per min per mg of protein at 30 degrees. Both showed similar apparent Km, values for each of the substrates in the aminoacylation reaction and the ATP-PPi exchange reaction.

• PMID: 4310598
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Reference Type

Comparative Study | Journal Article

Authors

Chlumecká V, Von Tigerstrom M, D'Obrenan P, Smith CJ

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