Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

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Abstract
E. gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hyroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose and blue dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 U/mg protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 .times. 10-6 M for L-leucine, 1.3 .times. 10-4 M for ATP and 1.3 .times. 10-6 M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a MW of 100,000. The amino acid composition of chloroplast leucyl-tRNA synthetase was determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.