Heat-stable translational inhibitor from rabbit reticulocyte lysates.

We have purified to apparent homogeneity a heat-stable (HS) factor from the postribosomal supernatant of rabbit reticulocyte lysates [(1988) FEBS Lett. 236, 479-483]. HS inhibits translation in hemin-supplemented lysates and induces phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 as does hemin deficiency. The translational inhibition produced by addition of HS to hemin-containing reticulocyte lysates and the accompanying phosphorylation of the eIF-2 alpha subunit can be prevented or reversed by NADPH generators including glucose 6-phosphate, NADPH itself, and also by dithiols, e.g., dithiothreitol, but not by fructose 1,6-bisphosphate or by monothiols, e.g., 2-mercaptoethanol. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS produces a pronounced increase of the HCI to proHCI ratio. It appeared possible that HS might be oxidized glutathione (GSSG) but this is not the case, for HS is not a substrate for highly purified glutathione reductase from rabbit erythrocytes. The spectral analysis of highly purified HS is consistent with the idea that HS could be a nucleotide derivative.

Purification of a novel heat-stable translational inhibitor from rabbit reticulocyte lysates.

We have purified to apparent homogeneity a novel heat-stable (HS) factor from postribosomal supernatants of rabbit reticulocyte lysates by heating for 10 min at 80 degrees C, fractionation on Sephadex, anion-exchange chromatography on QMA Accell, and gel filtration HPLC. The apparent molecular mass of HS is 500-1000 Da on the basis of its behaviour on gel filtration. Like a factor from bovine heart [(1982) Proc. Natl. Acad. Sci. USA 79, 3134-3137], the reticulocyte HS inhibits translation in hemin-supplemented lysates with biphasic kinetics similar to hemin deficiency and promotes phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF-2. It is active at nanomolar concentrations. Reticulocyte HS appears to be neither a peptide nor an oligonucleotide since HS activity was insensitive to proteolytic or nucleolytic digestion.