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The leucyl-tRNA and lysyl-tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl-agarose and purified to homogeneity. Conservation of activities during the purification required the presence of Triton X-100. The homogeneous enzymes corresponded to a monomer of Mr 129000 and a dimer of Mr 2 X 79000, respectively. Both were strongly adsorbed to the hydrophobic support phenyl-Sepharose, in conditions where the corresponding purified enzymes from yeast and Escherichia coli were not bound. Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl-tRNA and lysyl-tRNA synthetases derived from the complex displayed affinity for polyanionic supports. It is shown that proteolytic conversion of lysyl-tRNA synthetase to a fully active dimer of Mr 2 X 64000, leads to loss of both the hydrophobic and the polyanion-binding properties. These results support the view that each subunit of lysyl-tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues. The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.

Using a three-step procedure designed to minimize the risks of proteolysis, high molecular weight complexes containing the same seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, lysine, arginine, glutamic acid, and glutamine were purified from sheep liver and spleen, as well as from rabbit reticulocytes and liver. The polypeptide composition of these complexes, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is characteristic of the animal species from which they are derived. The complexes from sheep liver and spleen display indistinguishable polypeptide patterns composed of 11 major components. Of the 10 common components which characterize the complexes of rabbit reticulocytes and liver, 4 are also shared by the complexes from sheep, while 6 have distinctly different electrophoretic mobilities. Furthermore, in the case of the complex from rabbit reticulocytes, it is shown that the enzyme and polypeptide composition of the complex is independent of the purification method employed. The isolation of high molecular weight complexes of identical aminoacyl-tRNA synthetase and polypeptide compositions from two cell types as radically different as rabbit reticulocytes and hepatocytes suggests that these multienzyme complexes do not arise as artifacts of preparation and supports the view that they reflect a structural organization existing within the cell.

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl-tRNA and lysyl-tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl-tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl-tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl-tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl-tRNA synthetases. The sedimentation behaviour of the aminoacyl-tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.

Cultured Chinese hamster ovary cells were subjected to amino acid restriction to examine its effects on the level of expression of the nine aminoacyl-tRNA synthetase components of the multienzyme complex which was previously characterized (Mirande, M., Le Corre, D., and Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289). Lowering the methionine concentration in the medium from 100 to 1 microM led to growth arrest, rapid deacylation of tRNAMet, and progressive 2-fold elevation of the methionyl-tRNA synthetase level, as assessed by specific activity measurements and immunotitration. The levels of the other eight aminoacyl-tRNA synthetases were not affected. Total methionine deprivation led to the additional derepression of the leucyl- and isoleucyl-tRNA synthetase components, whereas the corresponding tRNAs remained fully acylated. These pleiotropic responses to total methionine restriction were abolished in the presence of 2 mM methioninol, suggesting that amino acid transport systems may play a role in the regulation of aminoacyl-tRNA synthetase expression. The effect of total deprivation of arginine, glutamine, isoleucine, leucine, lysine, or proline from the culture medium on the level of expression of the corresponding aminoacyl-tRNA synthetases was also examined. In all cases, no elevation of the level of the corresponding synthetase was observed. The behavior of methionyl-tRNA synthetase from Chinese hamster ovary cells displaying a 2-fold increased level of the enzyme due to methionine restriction was examined in detail. Failure to detect a free form of the enzyme by gel filtration, as well as the finding that the isolated complex displayed twice the amount of methionyl-tRNA synthetase relative to the other components, indicates that this multienzyme structure can accommodate at least one additional copy of one of its components.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).