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When rabbit reticulocyte lysates were gel-filtered on Sephadex (3-25 or (3-50, the rate of protein synthesis in the gel-filtered lysate was the same as in the parent lysate provided appropriate concentrations of ATP, G T P and spermidine or spermine were added. The polyamines were found to increase the rate of synthesis and to lower the Mg2 + optimum. Although gel-filtered lysates prepared in this way syntheses protein at a high initial rate, this rate is maintained for only about 20 min, after which it declines rapidly to a very low rate, even though optimal concentrations of haemin are present. This shut-off was completely prevented by the addition of low concentrations of sugar phosphates capable of acting as NADPH generators: glucose 6-phosphate, 2-deoxyglucose 6-phosphate, ribulose 5-phosphate and ribose 5-phosphate. Although the addition of isocitrate resulted in the generation of NADPH, the stimulation of protein synthesis was normally less than was achieved with glucose 6-phosphate. Dithiothreitol also promoted only partial activation, as did fructose 1,6-bisphosphate, a sugar phosphate which does not generate NADPH in this system. Full stimulation was observed, however, when both fructose 1,6-bisphosphate and either dithiothreitol or isocitrate were added. It is argued that the maintenance of maximum rates of protein synthesis in gel-filtered lysates requires the presence of both a sugar phosphate and a reducing agent, which can be dithiothreitol or an NADPH-generating system. Low concentrations of thioredoxin were shown to be required for the stimulatory effect of NADPH-generating systems, but not for stimulation by dithiothreitol. On the basis of these studies, a recommended procedure is given for the preparation of highly active gel-filtered lysates and gel-filtered nuclease-treated lysates.The rabbit reticulocyte lysate is unique amongst mammalian cell-free systems in that it synthesises protein at the same rate as the intact cell for periods of 60 min or more [I]. Almost all the work that has been done with this system has used crude undialysed lysates for the reason that dialysis or gel-filtration generally results in very considerable loss of translation activity. For many years this has been a challenging problem, since the ability to make gel-filtered lysates as active as the parent lysate would not only extend the utility of the lysate as a system for translation of heterologous mRNA [2], but would also allow one to define what low-molecular-weight cofactors are required to maintain a protein synthesis rate comparable to that in the intact cell. In fact, we and others have discovered some conditions which allow gel-filtered lysates to synthesise protein at high rates [3-91, but the exact nature of the requirements for low-molecular-weight cofactors, and the reasons underlying these requirements have remained obscure and controversial. In this and the following papers we describe recent work which clarifies and largely solves these outstanding problems.Although the data are extensive and the ar...

The study of gel-filtered rabbit reticulocyte lysates and lysates which have been passed through 2'5'ADPSepharose columns has shown that the presence of a sugar phosphate and a reducing system is necessary to maintain maximum rates of protein synthesis and to prevent an early inhibition. In this paper it is shown that the inhibition is duc to a reduced rate of initiation of translation and a decrease in the level ofmethionyl-tRNAf . 40S-ribosomal-subunit complexes. Sugar phosphates and reducing agents are shown to act co-operatively to prevent these changes: the absence of either sugar phosphates or reducing agents leads to a decrease in polysomes and in methionyl-tRNA . 40s-subunit complexes. The rcquirements for reducing power can be satisfied by either dithiothreitol or by an NADPH-generating system together with a functional thioredoxin/thioredoxin reductase system ; evidence is presented to show that these are required for the reduction of disulphide bonds. Incubation of lysates in the absence of a suitable reducing system leads to disulphide bond formation in a limited number of proteins prcsent in the lysate, but no disulphide bonds could bc detected in initiation factor eIF-2, the protein which catalyses the formation of methionyl-tRNA . 40s-subunit complexes. On the other hand, when these lysates were incubated undcr conditions in which protein synthesis is inhibited, eIF-2 was phosphorylated in the smallest of its three polypeptide chains (eIF-2%). The phosphorylation of elF-2a is shown to be controlled by the presence or absence of a suitable reducing system but not by sugar phosphates; it appears to be caused by activation of a protein kinase rather than through regulation of the rate of dephosphorylation of this protein. It is concluded that sugar phosphates d o not control the phosphorylation of eIF-2 but play soinc role as an activating cofactor affecting the rate of initiation of protein synthesis. The presence of a suitable reducing system is required to prevent or reverse disulphide bond formation in some critical protein(s) in the lysate; the oxidation of sulphydryl groups in this protein leads to activation of an eIF-2 kinase and hence to inhibition of initiation.In the preceding papers we have examined the properties of gel-filtered rabbit reticulocyte lysates and lysates which have been passed through 2'5'ADP-Sepharose affinity columns and we have shown that the presence of both sugar phosphates and a suitable reducing system is required to maintain high rates of protein synthesis for long incubation periods [l. 21. In this paper we are concerned with the mechanism by which these two agents prevent inhibition of protein synthesis. Inhibition of protein synthesis in gel-filtered lysates can also be prevented by the addition of high concentrations of GTP, or CAMP, or initiation factor eIF-2 [I, 3 -51, all of which have previously been found to be capable of overcoming the inhibition in normal (not gel-filtered) lysates incubated in the absence of haernin [6,7]. The nature of the lesion in lys...

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-offcould 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 lowmolecular-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-bisphosphate, 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 Esclieuicliiu 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.In the previous paper we showed that the rate of protein synthesis in gel-filtered rabbit reticulocyte lysates declined after about 20 min of incubation even though optimal concentrations of haemin were present [I]. Certain low-molecularweight compounds such as glucose 6-phosphate, or fructose 1,6-bisphosphate in combination with dithiothreitol, were found to prevent this shut-off. It was argued that the behaviour of the system was most readily interpretable in terms of a dual requirement, for a sugar phosphate and for a reducing agent, which could be either dithiothreitol or an NADPH-generating system. The difficulty in proving this dual requirement hypothesis is that when glucose 6-phosphate, the most effective activator of protein synthesis in gel-filtered lysates, is added both preconditions are automatically met: a sugar phosphate is present and NADPH is generated through glucose-6-phosphate dehydrogenase. In order to prove that glucose 6-