Tyrosyl-tRNA Synthetase from Baker's Yeast. Rapid Isolation by Affinity Elution, Molecular Weight of the Enzyme, and Determination of Essential Sulfhydryl Groups

Abstract

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 tRNA^Tyr. The homogeneous enzyme exhibits a molecular weight of 40000 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 100000 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 rapidly reacting sulfhydryl groups per subunit of molecular weight 40000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes. In addition an efficient purification method is described for isolating tRNA^Tyr from soluble ribo-nucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.