The plant aminoacyl-tRNA synthetases. Purification and characterization of valyl-tRNA, tryptophanyl-tRNA and seryl-tRNA synthetases from yellow-lupin seeds

Eur J Biochem. 1975 Mar 17;52(2):301-10. doi: 10.1111/j.1432-1033.1975.tb03998.x.

Abstract

Valyl-tRNA, tryptophanyl-tRNA, and seryl-tRNA synthetases from yellow lupin seeds Lupinus luteus were purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on aminohexyl-Sepharose column and affinity chromatography on tRNA-Sepharose column. Valyl-tRNA synthetase consists of one polypeptide chain of molecular weight 125000 as judged by Sephadex G-200 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent. Seryl-tRNA synthetase, Mr equals 110000, is composed of two 55000-Mr subunits. Tryptophanyl-tRNA synthetase exhibits molecular weight of 200000 on Sephadex G-200 and 37000 in dodecylsulfate-polyacrylamide gel electrophoresis. This indicates that tryptophanyl-tRNA synthetase consists of several subunits (probably four). Since the seryl-tRNA synthetase exhibits the same mobility on dodecylsulfate-polyacrylamide gels both in the presence and absence of reducing agent it is concluded that there is no covalent bond(s) between the subunits of the enzyme. There is also no covalent bond(s) between the subunits of tryptophanyl-tRNA synthetase. Effect of anti-sulfhydryl reagents, monovalent salts, pH and different buffers on activity of the three synthetases is described. Kinetic constants for the substrates of the synthetases are also given. dATP is a substrate for seryl-tRNA synthetase but not for valyl-tRNA and tryptophanyl-tRNA synthetases.

MeSH terms

  • Amino Acyl-tRNA Synthetases / isolation & purification*
  • Amino Acyl-tRNA Synthetases / metabolism
  • Chromatography, Affinity
  • Ethylmaleimide / pharmacology
  • Hydrogen-Ion Concentration
  • Hydroxymercuribenzoates / pharmacology
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight
  • Potassium Chloride / pharmacology
  • Seeds / enzymology*
  • Serine-tRNA Ligase / isolation & purification*
  • Tryptophan-tRNA Ligase / isolation & purification*
  • Valine-tRNA Ligase / isolation & purification*

Substances

  • Hydroxymercuribenzoates
  • Macromolecular Substances
  • Potassium Chloride
  • Amino Acyl-tRNA Synthetases
  • Serine-tRNA Ligase
  • Tryptophan-tRNA Ligase
  • Valine-tRNA Ligase
  • Ethylmaleimide