The Subunit Structure of Tryptophanyl Transfer Ribonucleic Acid Synthetase from Beef Pancreas

Abstract

Abstract The subunit structure of tryptophanyl-tRNA synthetase from beef pancreas (mol wt 108,000) has been studied by high speed and low speed equilibrium sedimentation, light scattering, gel electrophoresis, and gel filtration. All of these methods give a molecular weight of 58,000 ± 5,000 for the protein after denaturation in 6 m guanidine hydrochloride or in sodium dodecyl sulfate. Gel electrophoresis at different pH levels, after denaturation of the enzyme by 8 m urea or after dissociation by alkylation or oxidation of all of the —SH groups of the protein, shows a single polypeptide component; these data indicate that the subunits have the same molecular weight and the same charge. Hydrolysis of the sodium dodecyl sulfate-denatured protein by carboxypeptidases A and B indicates that the COOH-terminal sequence should be -Leu-Phe-Gln; these amino acids are liberated in a ratio close to 2 eq per molecule of native enzyme. By a tritium labeling method only one type of COOH-terminal residue is detected. No free NH2-terminal amino group can be detected. Gel electrophoresis and NH2-terminal group analysis of the cyanogen bromide digestion products and fingerprints of the tryptic peptides of the enzyme are in agreement with a chemical identity of the polypeptide chains obtained after denaturation. Since it has been shown that tryptophanyl-tRNA synthetase can bind 2 molecules of substrate (tryptophan or tryptophanyl adenylate), and since no polypeptide chain of molecular weight smaller than half of that of the native enzyme could be obtained in any denaturing condition, it is concluded that this amino acid activating enzyme in its native state is a dimer made up of 2 identical subunits.