Cytochalasin A and B

Abstract

Isoleucyl-tRNAXynthetase Catalyzed Deacylation of Val-tRNAI'"Vol. 247, No. 9 synthetase-Val-AMP complex in the reaction mixture.In the case of the Val-tRNAne deacylation reaction, a a-fold increase in the concentration of isoleucyl-tRNA synthetase led to no change in the rate of deacylation.These data, therefore, support the hypothesis that Val-tRNAne is an intermediate in the tRNAm-induced hydrolysis of Val-AMP.This possibility seems even more likely when, as mentioned above, it is taken into account that any modification of tRNAne which destroys its amino acid acceptor ability also destroys its ability to induce hydrolysis of Val-AMP(1).Thus, the data of Fig. 3 would suggest that the isoleucyl-tRNA synthetase-Val-AMP complex reacts with tRNAne in a relatively slow step to produce Val-tRNAne which is then immediately deacylated in a rapid step.Some caution in interpretation must be exerted because it was found that the rate of Val-AMP hydrolysis is unaffected by 2 mM EDTA (with no added Mg2+), but the isoleucyl-tRNA synthetase-catalyzed deacylation of Val-tRNAne is essentially abolished under the same conditions.However, EDTA does not quantitatively remove Mg2+ at pH 7, and it is quite possible that small amounts of non-EDTA-bound Mg2f were present in the Val-AMP assay system.It was found that in the absence of EDTA only small amounts of Mg2+ (~10-6 to lo+ M) are necessary to give a deacylation of Val-tRNAne which is more rapid than the hydrolysis of Val-AMP.In conclusion, the present study strongly supports the previously advanced hypothesis that the weak deacylase activity which isoleucyl-tRNA synthetase exhibits toward Ile-tRNAne should be greatly enhanced with Val-tRNAm as the substrate for the deacylation (3).It is quite possible that this activity plays a physiological role i n viva.Moreover, the comparative rate data given in Fig. 3 gives some support to the hypothesis that Val-tRNAne is a transient intermediate in the tRNAneinduced hydrolysis of isoleucyl-tRNA synthetase-bound Val-AMP.