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 [P] EC 6.1.1.b) from rat liver has been purified 1500 fold by the addition of hydroxylapatite chromatography to a previously developed procedure. From sodium dodecyl sulfate electrophoresis the enzyme appears to be composed of two types of subunits with molecular weights of 74,500 and 69,000, respectively. Thermal inactivation appears to be associated with breakdown of the larger subunit. The effect of various ligands on the thermal inactivation of the enzyme was investigated. Triphosphates, in general, had a destabilizing effect with adenosine triphosphate (ATP) exerting the greatest influence on the rate of thermal decay of enzyme activity. It seems that the pyrophosphate moiety is responsible for the labilization in these cases since, under the same conditions, sodium pyrophosphate had an even greater effect on the thermal stability of the enzyme than ATP. The presence of magnesium ions reduced the effect of both of these substrates. In the presence of magnesium ion, phenylalanine protected against heat inactivation, but its thermal effect was additive with that of ATP, providing no evidence for the formation of an aminoacyl adenylate. Transfer RNA(Phe) from rat liver and yeast also protected the enzyme against heat inactivation both in the absence and presence of magnesium. Studies with tRNA(Val), tRNA(fMet), tRNA(Arg), and tRNA(Phe) from Escherichia coli indicated that the amount of protection a tRNA afforded against heat inactivation was reflected in the extent to which it was chargeable by the enzyme, while those tRNAs which were not chargeable but still were bound to the enzyme seemed to strain the enzyme, thus increasing its heat lability.