Abstract
Abstract 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 µmole 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 µmoles of inorganic pyrophosphate exchanged per min per mg of protein at 30°. Both showed similar apparent Km values for each of the substrates in the aminoacylation reaction and the ATP-PPi exchange reaction.
Highlights
L-amino acids were purchased from Calbiochem, and 14C-amino ; Lys-tRNA, lysyl-tRNA; lysinespecific tRNA covalently linked to lysine; Asso unit, that amount of RNA which, when dissolved in 0.05 M Tris-HCl buffer, pH 7.4, 25”, in a total volume of 1 ml, shows an optical density of 1.0 measured at 260 m in a l-cm light path cuvette
The aminoacylation reaction very similar to those reported for Assays were performed at 30” in Tris-HCl buffer by the pro- similar enzymes obtained in homogeneousform from other cedures described in the text
This tions of 3 to 80.pg of enzyme per ml with 4 to 12As@ units of is apparently the first observation of the phenomenonwith a tRNA per ml), the major componentcausedacylation of 5.0% of purified Lys-tRNA synthetase,there are a numberof reports of the chainsof tRNA, whereasthe sameconcentrationof the minor the isolation of multiple formsof other aminoacyl-tRNA synthecomponentcharged3.5 to 4.5% of the tRNA with the sameassay tases (2630)
Summary
Lysyl transfer ribonucleic acid synthetase (L-lysine: tRNA ligase (AMP), EC 6.1.1.6) was pursed to a state of apparent homogeneity from bakers’ yeast. 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. The two protein componentswere 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 azPpyrophosphate into ATP in the absence of lysine-acceptor RNA, each showing the same specific activity value of approximately 3 rmoles of inorganic pyrophosphate exchanged per min per mg of protein at 30’. Lagerkvist and Waldenstriim (7) have achieved purification of the synthetase from early log phase yeast cells but were unsuccessful in attempts to prepare the enzyme from commercial yeast.[2]
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