Tryptophanyl Transfer Ribonucleic Acid Synthetase from Bovine Pancreas: II. The chemically different subunits

Abstract

Abstract Bovine pancreas tryptophanyl transfer RNA synthetase, a crystalline enzyme, is shown by filtration on a carefully calibrated Sephadex G-150 column and by equilibrium centrifugation to have a molecular weight of 110,000 (s20,w = 5.6) in the presence of 10-4 M tryptophan. When the enzyme is treated with 4 M guanidine-hydrochloride for several days or with 8 M urea, values for the s20,w become 1.6 and 2.0, respectively. The enzyme dissociates slowly (to s20,w = 3.0) on dialysis against Tris-glycine buffer, pH 8.5, or 0.1 M ammonium bicarbonate, pH 8.8. A very slow dissociation is caused by 0.2 M β-mercaptoethanol which is rapidly speeded up on the addition of 1% sodium dodecyl sulfate. Titration of native enzyme with 5,5'-dithiobis(2-nitrobenzoate) indicates the presence of four free —SH groups; titration in the presence of 8 M urea indicates six —SH groups; titration of sodium borohydride-reduced enzyme indicates 14 —SH groups per molecule of 110,000 molecular weight. The enzyme has been reduced with 0.2 M β-mercaptoethanol in 8 M urea and carboxymethylated with 14C-iodoacetic acid. The carboxymethylated protein is eluted as a single peak in a position corresponding to a particle with a molecular weight of 27,000 to 30,000 from a Sephadex G-150 column. Sedimentation equilibrium experiments show a single molecular weight species of 27,000 (s20,w = 1.9). However, chromatography on carboxymethyl-Sephadex separates the carboxymethylated protein into two distinct radioactive protein peaks. The carboxymethylated proteins were acetylated, digested with trypsin, and subjected to peptide mapping. A total of seven 14C-cysteine peptides and 22 arginine-containing peptides was identified in both protein species, while the amino acid composition of the total unfractionated carboxymethylated protein shows 14 carboxymethylcysteine residues and 44 arginine residues per molecule of 110,000 molecular weight. The amino acid compositions of the two separated carboxymethylated proteins show significant differences, one carrying 6 half-cysteine residues, the other only 1. Catalytic hydrazinolysis of the individual carboxymethylated proteins gives 1 mole of glutamic acid and 1 mole of glycine, respectively, per 27,000 molecular weight. These experiments lead to the conclusion that a molecule of tryptophanyl-tRNA synthetase is made up of four subunits per molecular weight of 110,000. These subunits consist of two kinds of monomers with different primary structures. Tryptophanyl-tRNA synthetase activity is not restricted to the tetrameric protein but is also found in a smaller molecular species (s20,w = 3.0 to 3.3). This molecule is found naturally in preparations of the enzyme and is also formed on dissociation of the tetrameric enzyme. These findings and those reported in the accompanying paper suggest that the catalytic form of the enzyme, the protomer, may be a dimer, and that the tetrameric form has no special functional significance and most likely is an artifact produced by the presence of tryptophan during the purification procedure.