Slow Diffusion of Glutamate and ATP-Mg into High-Molecular-Weight Complexes Containing the Glutamyl-tRNA Synthetase from Bovine Brain

The glutamyl-tRNA synthetase partially purified from bovine brain is associated to complexes of molecular weight higher than 106, and catalyzes the formation of glutamyl-tRNA at an initial rate which is much slower than the final constant rate reached after about 5 min. However, the aminoacylation starts at this constant high rate if and only if the enzyme preparation has been preincubated with glutamate, ATP and Mg2+. Following a preincubation of these complexes with γ-methyl-L-glutamate, a competitive inhibitor of the glutamyl-tRNA synthetase, the initial rate of formation of glutamyl-tRNA is much slower than following a preincubation with glutamate and the inhibitor, but reaches the same constant value. Moreover, if the enzyme has been preincubated with glutamate, the rate of aminoacylation decreases only a few minutes after the addition of the inhibitor. These results and those obtained after the sequential addition of unlabeled glutamate and [14C]glutamate in an aminoacylation reaction rule out the possibility of a slow ‘activation’ of the enzyme by its substrates glutamate and ATP-Mg2+. Finally, the association of glutamate with these high-molecular-weight complexes has been detected by filtration on nitrocellulose discs. We have observed similar kinetic patterns with the glutaminyl-tRNA synthetase, but not with seven other aminoacyl-tRNA synthetases. The results indicate that glutamate, glutamine and ATP-Mg2+ must diffuse relatively slowly inside the high-molecular-weight complexes before reaching their specific binding sites on the corresponding aminoacyl-tRNA synthetases. The physiological significance of these results is discussed in the context of the important metabolic and neurotransmitter functions of glutamate and glutamine in the brain.