Studies on rat liver phenylalanyl transfer ribonucleic acid synthetase. II. Further purification, substrate specificity, and effects of substrates on heat inactivation.

The phenylalanyl-tRNA synthetase (L-phenylalanine: tRNA ligase [AMP] EC 6.1.1.6) from rat liver was purified SOO-fold by phosphocellulose and Sephadex G-100 chromatography. The behavior of this enzyme during purification suggests that intracellularly it exists as a complex with its specific transfer ribonucleic acid (tRNAPh”). The sedimentation coefficient in sucrose gradients of the purified enzyme is 7.7 S while that of the complex with yeast tRNAPh” is 11.6 S. This change in sedimentation coefficient is most compatible with the binding of 4 tRNAPh” molecules to a synthetase of about 180,000 molecular weight although alternative explanations cannot be fully discounted. Similar changes in sedimentation coefficient were observed on binding rat liver tRNAPh” and yeast tRNA Phe from which the Y base had been removed. Heat inactivation of the synthetase, as measured by its decrease in ability to aminoacylate yeast tRNAPhe, proceeds with apparent first order kinetics between pH 6 and pH 8. At pH 6.3 the rate constant for inactivation, kc, increases markedly with increasing ionic strength. However, at pH 7.5, ki decreases somewhat with increasing ionic strength. An inflection point in the plot of ki in 0.2 M KC1 uersus pH occurs at pH 6.4 and corresponds to the known pK of histidyl residues. Glycerol substantially protects against heat inactivation under all conditions of ionic strength and pH examined.