The High-Temperature Inactivation of Rabbit Reticulocyte Lysates by a Haemin-Independent Mechanism

Abstract

Exposure of rabbit reticulocytes or their lysates to 42–45 °C leads to a decrease in the rate of polypeptide chain initiation which resembles in several respects the lesion produced in reticulocyte lysates incubated in the absence of haemin. The aim of the present investigation was to establish whether the two processes were mediated by a similar mechanism, i.e. by a haemin-controlled, catalytically active, soluble inhibitor. It was found that the addition of haemin during exposure to elevated temperatures cannot prevent nor rescue the high-temperature-dependent inactivation of reticulocytes or their lysates. Assay systems which can detect the haemin-controlled repressor revealed that supraoptimally heated lysates and the postribosomal supernatants derived from them contain a soluble inhibitor. This inhibitor, although forming in the presence of haemin, resembles in several properties the haemin-controlled repressor. However, it appears to fractionate differently with ammonium sulphate and to decay after prolonged supraoptimal heating of either cells or lysates (but not of postribosomal supernatant). Moreover the 0.5 M KCl ribosomal wash obtained from heated cells or lysates was as active as preparations from control cells in supporting protein synthesis at 30–37 °C in fresh or preheated lysates and in fractionated cell-free systems synthesizing globin de novo. These results suggest that the lesion in polypeptide chain initiation produced in reticulocyte lysates by elevated temperatures or by incubation in the absence of haemin may be mediated by similar but not necessarily identical mechanisms.