Macromolecular Complexes of Aminoacyl‐tRNA Synthetases from Eukaryotes

The gel-filtration behaviour of a high-molecular-weight complex containing seven aminoacyl-tRNA synthetases purified from sheep liver was examined on columns of 6% agarose (Bio-Gel A-5m). Evidence is provided for selective interaction of the complex with the agarose matrix. The binding capacity of agarose for the complex is dictated by the ionic strength of the equilibrating buffer: it is low but significant in 25 mM potassium phosphate buffer at pH 7.5 (ionic strength 0.068 M) and is considerably enhanced in 50 mM Tris-HCl buffer at the same pH. In either case, raising the salt concentration leads to nearly complete recovery of enzyme activities. In a column equilibrated in 25 mM phosphate buffer, application of the complex at a protein concentration in large excess over the binding capacity of the gel leads to co-elution of each of the seven aminoacyl-tRNA synthetases as a unique, symmetrical peak of apparent molecular weight close to 106, with 80%, recovery of activities. Conversely, application of the complex at low protein concentration, in amounts equivalent to the binding capacity of the gel, leads to complete retention of the enzymes which may be recovered by raising the phosphate concentration to 0.2 M. It is further shown that application of the complex at low protein concentration on a column equilibrated with 0.2 M potassium phosphate buffer to prevent interaction with the matrix, leads to co-elution of each of the aminoacyl-tRNA synthetases as a unique peak of apparent molecular weight close to 106. This result attests to the remarkable stability of the complex, which fails to dissociate at high salt concentration, even in the diluted state. The above results are best interpreted as the reflection of ion-exchange-mediated interactions between the residual charged group on the agarose gel and the components of the high-molecular-weight complex. The preferential retention of the complex on agarose in ionic conditions (i. e. 25 mM phosphate or 50 mM Tris-HCl buffers at pH 7.5) which ensure normal gel filtration behaviour of several marker proteins, may be ascribed to tighter binding of the complex due to multiple-site interactions on account of its larger size.