Molecular Forms of the Chloroplast Leucyl-tRNA Synthetase of Euglena gracilis Z Strain

Abstract

Summary The method of separation by non-denaturing polyacrylamide gel electrophoresis and determination of enzyme activities in gel slices has been used to discriminate 2 molecular forms of the chloroplast-specific leucyl-tRNA synthetase in Euglena gracilis Z strain. The purified enzyme shows a molecular mass of 105kDa by gel filtration but appears to consist of a pseudodimerform (α 2 ') of 53 kDa that is likewise enzymatically active. This form (II) correlates with R M 0.42 in non-denaturing gel electrophoresis ( p H 8.3). It is likewise observed in isolated chloroplasts where it predominates a larger molecular form (I) with a migration ratio of R M 0.20. We were unable to prevent the occurrence of form II by addition of various proteinase inhibitors during cell homogenization or by variation of the method and duration of enzyme preparation. Chloroplast leucyl-tRNA synthetase with high enzyme activity has been synthesized by a RNA-programmed cell-free wheat germs. This in vitro translation product co-migrates mainly with form I, but active form II is always present in a minor proportion of ca. 10 %. It is suggested that our E. gracilis Z strain contains a monomeric chloroplast leucyl-tRNA synthetase with a putative intramolecular nick that causes the observed pseudodimeric form.