Isolierung und Charakterisierung der Seryl- und Phenylalanyl-tRNA-Synthetase aus Hefe

Abstract

A procedure for the simultaneous isolation of seryl- and phenylalanyl-tRNA synthetase (EC 6.1.1.11 and EC 6.1.1.20) from yeast is described. Some other synthetases as well as tRNA nucleotidyltransferase can also be obtained in an enriched state. The isolated seryl- and phenylalanyl-tRNA synthetases were compared to earlier preparations with respect to purity, specific activity and structure. Previous investigations with fluorescence spectroscopy and kinetic methods were complemented and extended by experiments on the specificity of aminoacylation and on the isolation, by sucrose gradient centrifugation, of complexes between synthetase and tRNA or tRNA fragments. A protection of synthetases against inactivation by addition of substrates was observed. The dissociation of seryl-tRNA synthetase, at low concentrations, into monomer subunits was investigated by chemical modification with bifunctional reagents and by kinetic experiments. By modification of SH-groups fluorescent dyes were incorporated into both, seryl- and phenylalanyl-tRNA synthetase which retained most of their activity. The binding of tRNAPhe to phenylalanyl-tRNA synthetase which had been modified with pyrene maleimide was followed by fluorescence intensity measurements.