Purification and properties of chloroplast leucyl-tRNA synthetase from a higher plant: Phaseolus vulgaris.
The chloroplast leucyl-tRNA synthetase from Phaseolus vulgaris was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite, and phosphocellulose. Finally, the pure enzyme was obtained after affinity chromatography on Blue Sepharose CL-6B using specific elution with a pure Escherichia coli tRNALeu isoacceptor. The specific activity of chloroplast leucyl-tRNA synthetase (1550 units/mg) is the highest ever obtained for a higher plant aminoacyl-tRNA synthetase. The purified enzyme has an optimal pH of 9.0 and Km values are, respectively, 2.6 X 10(-6) M for unfractionated E. coli tRNA, 0.85 X 10(-6) M for E. coli tRNA5Leu, 1.8 X 10(-4) M for ATP, and 1.4 X 10(-5) M for L-leucine. Chloroplast leucyl-tRNA synthetase is a large monomer which has a Mr of 122,000 as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate and urea and by gel filtration. Determination of Stokes radius, diffusion coefficient, and frictional ratio suggests that the enzyme structure is rather compact. The amino acid composition shows a relatively large proportion of apolar residues. Specific antibodies were raised in rabbits against the pure chloroplast leucyl-tRNA synthetase.
D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D002736:Chloroplasts / Q000201:enzymology*; D002845:Chromatography; D004355:Drug Stability; D004926:Escherichia coli / Q000032:analysis; D007887:Fabaceae; D007935:Leucine-tRNA Ligase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010946:Plants, Medicinal; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism
D000596:Amino Acids; D012346:RNA, Transfer, Amino Acyl; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase