The Use of Affinity Chromatography on 2′5′ ADP‐Sepharose Reveals a Requirement for NADPH, Thioredoxin and Thioredoxin Reductase for the Maintenance of High Protein Synthesis Activity in Rabbit Reticulocyte Lysates

Rabbit reticulocyte lysates were passed through 2′5′ ADP‐Sepharose columns under conditions in which the gelfiltration effect was negligible and low‐molecular‐weight compounds were retained in the flow‐through lysate. Glucose‐6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase and glutathione reductase were quantitatively adsorbed by the column and removed from the lysate, but isocitrate dehydrogenase and thioredoxin reductase were retained in the flow‐through lysate. The initial rate of protein synthesis in lysates treated in this way was normal, but synthesis stopped after about 20 min of incubation. This shut‐off could be prevented by the addition of dithiothreitol or by providing a means of NADPH generation, which could be achieved either by adding isocitrate or glucose‐6‐phosphate dehydrogenase. Further experiments used lysates which were first gel‐filtered to remove low‐molecular‐weight metabolites and then passed through 2′5′ ADP‐Sepharose columns. Under these conditions thioredoxin reductase was efficiently adsorbed by the affinity column, in addition to the three enzymes already listed. The maintenance of full protein synthesis activity in these lysates required the addition of both a sugar phosphate and a reducing agent. The sugar phosphate requirement could be satisfied by glucose 6‐phosphate, or 2‐deoxyglucose 6‐phosphate, or fructose 1,6‐biphosphate, but not by 6‐phosphogluconic acid. The requirement for reducing agent could be met by the addition of dithiothreitol, or by an NADPH‐generating system together with rabbit thioredoxin reductase. Purified thioredoxin reductase from Escherichia coli was also effective provided E. coli thioredoxin was also added, but the addition of glutathione with glutathione reductase did not activate protein synthesis. It is concluded that there is a dual requirement for the maintenance of high rates of protein synthesis in reticulocyte lysates: certain sugar phosphates must be present, in addition to an NADPH‐generating system and a functional thioredoxin/thioredoxin reductase system.