The Aminoacyl Ribonucleic Acid Synthetases

Abstract

Abstract Threonyl ribonucleic acid synthetase was purified 400-fold from rat liver. During stepwise chromatography on diethylaminoethyl cellulose, two fractions with this enzymatic activity were observed. The separated fractions give distinct elution patterns on rechromatography on the same ion exchanger. Threonyladenylate-enzyme complex prepared with 14C-threonine and 3H-adenosine triphosphate shows a stoichiometry of 1:0.96 of threonine to adenosine 5'-phosphate. Threonyl transfer from the enzyme-bound complex to soluble RNA is inhibited slightly by AMP but strongly inhibited by pyrophosphate and by the combination of AMP and pyrophosphate. The transfer reaction is completely inhibited by ethylenediaminetetraacetate but this inhibition can be overcome by addition of an excess of magnesium. The amino acid is transferred very rapidly, achieving complete transfer in less than 2 min at 37° and in 12 min at 0°. The threonyladenylate-enzyme complex can be chromatographed in active form on DEAE-cellulose ion exchanger. At pH 7.0 and 37°, the half-life of the complex is approximately of 7 min and its rate of inactivation coincides with the hydrolysis of synthetic threonyladenylate. At pH 4.5, the half-life is of approximately 4 min and is determined by the inactivation of the enzyme moiety. Threonyl-RNA synthetase can become labeled by incubation with radioactive ATP and magnesium in the absence of added threonine.