A BROADLY APPLICABLE CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR MEASURING AMINOACYL-TRANSFER-RNA SYNTHETASE-ACTIVITY

We describe a convenient, simple and novel continuous spectrophotometr ic method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate gene rated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphat e, which in turn is used as the substrate of purine nucleoside phospho rylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-m ethylpurine ribonucleoside, Of the reaction products, ribose 1-phospha te and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorb ance at 360 nm relative to the nucleoside and hence provides a spectro photometric signal that can be continuously followed. The nondestructi ve nature of the spectrophotometric assay allowed the re-use of the tR NAs in question in successive experiments. The usefulness of this meth od was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptop hanyl-tRNA synthetase, Initial velocities measured using this assay co rrelate closely with those assayed by quantitation of [H-3]Gln-tRNA or [C-14]Trp-tRNA formation respectively. In both cases amino acid trans fer from the aminoacyl adenylate to the tRNA represents the rate deter mining step. In addition, aminoacyl adenylate formation by aspartyl-tR NA synthetase was followed and provided a more sensitive means of acti ve site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.