MACROMOLECULAR COMPLEXES FROM SHEEP AND RABBIT CONTAINING 7 AMINOACYL-TRANSFER RNA-SYNTHETASES .2. STRUCTURAL CHARACTERIZATION OF THE POLYPEPTIDE COMPONENTS AND IMMUNOLOGICAL IDENTIFICATION OF THE METHIONYL-TRANSFER RNA-SYNTHETASE SUBUNIT

The extensively purified multienzyme complexes from sheep and rabbit livers containing 7 aminoacyl-tRNA synthetases specific for Ile, Leu, Met, Gln, Glu, Lys, and Arg displayed characteristic 1-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic [PAGE] patterns composed of 11 and 10 major polypeptide components, respectively. Their polypeptide compositions revealed by 2-dimensional electrophoresis, including isoelectric focusing in 9 M urea, were not significantly more complex. The isoelectric point of each component from the 2 complexes fell within the pH range of 6.2-7.1, with the notable exception of the common polypeptide of MW = 43,000, which was distinctly basic. The apparent MW of each component from both complexes was determined by SDS-PAGE. Four polypeptides, corresponding to MW of 139,000, 129,000, 43,000 and 38,000, were common to both complexes. The other components from the 2 complexes displayed similar yet clearly distinct MW. 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 1 copy. Following SDS-PAGE, 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 MW = 103,000 and 108,000, respectively. Mild trypsin treatment of the 2 native complexes generated fully active forms of methionyl-tRNA synthetase, with MW of 68,000 and 69,500, respectively. The kinetics of proteolysis showed that modification proceeded sequentially through discrete intermediates.