Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. II. Structural characterization of the polypeptide components and immunological identification of the methionyl-tRNA synthetase subunit.

The extensively purified multienzyme complexes from sheep and rabbit livers containing seven aminoacyl-tRNA synthetases specific for Ile, Leu, Met, Gln, Glu, Lys, and Arg displayed characteristic one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic patterns composed of 11 and 10 major polypeptide components, respectively. Their polypeptide compositions revealed by two-dimensional electrophoresis, including isoelectric focusing in 9 M urea, were not significantly more complex. The isoelectric point of each component from the two complexes fell within the pH range of 6.2 to 7.1, with the notable exception of the common polypeptide of Mr = 43,000 which was distinctly basic. The apparent molecular weight of each component from both complexes was determined by SDS-polyacrylamide gel electrophoresis. Four polypeptides, corresponding to molecular weights of 139,000, 129,000, 43,000, and 38,000 were common to both complexes. The other components from the two complexes displayed similar yet clearly distinct molecular weights. The molar ratios of the polypeptides, estimated by densitometry scanning of stained SDS-polyacrylamide gels, indicated that several components from each complex may be present as more than one copy. Following SDS-polyacrylamide gel electrophoresis, the methionyl-tRNA synthetase component from each complex was identified by the protein blotting procedure, using specific antibodies and 125I-labeled protein A. The unique labeled bands from the complexes of sheep and rabbit precisely matched the major polypeptides of Mr = 103,000 and 108,000, respectively. Mild trypsin treatment of the two native complexes generated fully active forms of methionyl-tRNA synthetase, with molecular weights of 68,000 and 69,500, respectively. The kinetics of proteolysis showed that modification proceeded sequentially through discrete intermediates.