PARTIAL PURIFICATION AND PROPERTIES OF PROLYL-RNA SYNTHETASE OF RAT LIVER

Abstract

Prolyl-RNA (prolyl ribonucleic acid) synthetase has been purified 30-fold from a 105,000 × g supernatant of a rat liver homogenate by precipitation at pH 5.0, heat treatment at 55 °C for 3.0 minutes in the presence of 1.0 mM ATP (adenosine triphosphate), and by ammonium sulphate fractionation. The enzyme catalyzed proline-dependent ATP-32PP (PP, inorganic pyrophosphate) exchange and the formation of prolyl hydroxamate and of prolyl-RNA. Although the enzyme did not catalyze the formation of hydroxyprolyl-RNA, it catalyzed a slight hydroxyproline-dependent ATP-32PP exchange and the formation of a small amount of hydroxyprolyl hydroxamate which was much less than the amount of prolyl hydroxamate formed under the same conditions. The enzyme is thus not quite specific for proline activation, but is specific for amino acyl-RNA formation. It is probably concerned in protein biosynthesis.In the proline-dependent ATP-32PP exchange reaction the enzyme showed optimum activity in the pH range 6.2–8.2 and no activity at pH 5.0. The apparent Kmfor proline in this reaction was found to be 0.43 mM. Mg++was required for activity. Prolyl-RNA formation was optimal at pH 8.0. The apparent Kmfor proline in this reaction was found to be 2.5 μM. The effects of some proline analogues on proline activation were studied. Hydroxyproline and thioproline were found to inhibit both proline-dependent ATP-32PP exchange and prolyl-RNA formation. Thioproline was a competitive inhibitor of the exchange reaction and showed a KIof 0.95 mM. Pyrrolidone carboxylate had no appreciable effect on proline activation.