Tyroslyl-tRNA synthetase from baker's yeast. Rapid isolation by affinity elution, molecular weight of the enzyme, and determination of essential sulfhydryl groups.
Tyrosyl-tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000. After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE-cellulose and chromatography on CM-Sephadex, the critical purification step is the elution of the cation-exchanger-bound tyrosyl-tRNA synthetase with tRNATyr. The homogeneous enzyme exhibits a molecular weight of 40 000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate-gel electrophoresis under reducing and non-reducing conditions. Gel filtration experiments show a molecular weight of about 100 000 indicating the existence of an active dimeric form. The possibility of proteolytic cleavage of the enzyme is excluded. The reaction of tyrosyl-tRNA synthetase with p-chloromercuribenzoate and N-ethylmaleimide reveals two repidly reacting sulfhydryl groups per subunit of molecular weight 40 000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes. In addition an efficient purification method is described for isolating tRNATyr from soluble ribonucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002846:Chromatography, Affinity; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014448:Tyrosine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism
D002729:Chloromercuribenzoates; D046911:Macromolecular Substances; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D005033:Ethylmaleimide