[Purification and properties of phenylalanyl-tRNA synthetase from baker's yeast].

Abstract

Abstract Purification and some properties of phenylananyl-tRNA synthetase from baker's yeast Phenylalanyl-tRNA synthetase has been isolated from baker's yeast with a 600-fold purification. The different steps of the preparation are: (NH 4 ) 2 SO 4 precipitation of the 78 000 × g crude extract (between 50 and 65 % saturation), chromatography on DEAE-cellulose, CM-Sephadex C-50 and hydroxylapatite. The enzyme appears to be homogeneous on hydroxylapatite chromatography, sucrose gradient centrifugation and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation or equilibrium sedimentation studies give an average value of 220 000. Amino acid composition has been determined. No end group can be detected by the dansyl method. In the presence of either ATP and phenylalanine or tRNA Phe , the number of free thiol groups titrated with DTNB decreases. The enzyme is dissociated by 8 M urea or 1 % sodium dodecyl sulphate into two different equimolar components. The molecular weights of these 2 components were estimated to be 56 000 and 63 000, respectively, by polyacrylamide gel electrophoresis. The results suggest that the enzyme has a 4-subunits structure A 2 B 2 . The kinetics of the PP i -ATP exchange and aminoacylation reactions of tRNA Phe have been determined.