the use of affinity chromatography on 2′5′ adp-sepharose reveals a requirement for nadph,...

Eur. J . Biochem. 131, 303-311 (1981) ’( FEBS 1983

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

Tim HUNT, Pamela HERBERT, Elizabeth A. CAMPBELL, Christos DELIDAKIS, and Kichard J . JACKSON

Department of Biochemistry, University of Cambridge

(Received August 31/December 6, 1982) - EJB 5952

Rabbit reticulocyte lysates were passed through 2’5’ADP-Sepharose columns under conditions in which the gel- filtration 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 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-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-molecular- weight 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 hy- pothesis 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- ____

Ahbwviutiuns. 2’5’ADP-Sepharose, adenosine 2’,5’-bisphosphate linked to Sepharosc at C-8 via a 6-aminohexyl group; CAMP, adenosine 3’,5’-monophosphate; Glc6P dehydrogenase, glucose-&phosphate dehydrogenase; GSH, glutathione; GSSG, oxidised glutathione: GSSG reductase, glutathione reductase; Nbs,, 5.5’-dithio-bis(2-nitrobenz[)ic acid).

Enzymes. Isocitrate dehydrogenase (EC 1.1.1.42); 6-phosphogluconate dehydrogenase (EC 1 . I . 1.44); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); glutathione reductase (EC 1.6.4.2): thioredoxin reductase (EC 1.6.4.5); creatine kinase (EC 2.7.3.2).

phosphate stimulates protein synthesis because it is fulfilling both requirements, it seems necessary to be able to study lysates which lacked glucose-6-phosphate dehydrogenase and therefore could not generate NADPH from metabolism ofGlc6P. In this paper we show that this objective can be achieved by passing the lysate through a column of 2’5’ADP-Sepharose, an affinity medium with high specificity for enzymes that use NADP or NADPH as cofactor [2,3]. The properties of Iysates treated in this way, and especially lysates that had been gel- filtered before passage through 2’5’ADP-Sepharose, confirmed the dual requirement for sugar phosphates and reducing agents. We show in this paper that the former requirement can be met by glucose 6-phosphate, 2-deoxyglucose 6-phosphate and fructose 1,6-bisphosphate, but not by 6-phosphogluconate; the latter requirement can be satisfied by either dithiothreitol or by an NADPH-generating system in conjunction with a thioredoxin/thioredoxin reductase system.

MATERIALS AND METHODS

Materials

DEAE-Sepharose CL 6 B and 2‘5‘ADP-Sepharose were obtained from Pharmacia. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate de-

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hydrogenase were from Boehringer, and bovine insulin was from Sigma. The sources of the other materials were as in the preceding paper [I]. Purified thioredoxin and thioredoxin reductase from Escherichiu coli were generous gifts from Dr A. Holmgren.

Preparation o f Lysutes a id Gel-Filtered Lysutes

Rabbit reticulocyte lysates were prepared as described in the preceding paper [I]. Gel-filtration of lysates was performed using Sephadex G-25 columns equilibrated in buffer A as described in this paper [I]. Lysates were made 20 pM in haemin and 50 pg/ml in creatine kinase before gel-filtration and the pooled material from the column was made 0.5 mM in ATP, 0.1 mM in GTP and 0.5 mM in spermidine, before freezing in liquid nitrogen in small aliquots.

Amino Acid Incorporation Assuys

In the standard assay [14C]valine (30 Ci/mol) was added at a final concentration of 84pM; 5 pl samples were taken to determine the incorporation, as described in the preceding paper [I]. In short-term assays of column fractions, ["S]methionine (1 200- 1 500 Ci/mmol) was added to give a final isotopic concentration of 0.1 mCi/ml; samples were taken after 10min of incubation as described in the preceding paper [I].

Bufrers

0.1 mM EDTA, 10 mM Hepes/KOH pH 7.2.

EDTA.

Buffer A: 25mM KC1, 10mM NaCI, l . l m M MgCl,,

Buffer B: 0.1 M potassium phosphate pH 7.5, 10mM

Enzyme assay.^

Glucose-6-phosphate dehydrogenase was assayed by adding enzyme or lysate to a solution of 0.2mM NADP and 0.5 mM Cilc6Pin buffer B, and following the increase in A, , , in a recording spectrophotometer. Glutathione reductase was assayed by adding enzyme to a solution of 0.5 mM GSSG and 0.2mM NADPH in buffer B and following the decrease in A,4o. Thioredoxin reductase was routinely assayed by its ability to catalyse the reduction of 5,5'-dithio-bis(2-nitro- benzoic acid) (Nbs,) 141. To 0.5 ml of a solution of 0.2 mM NADPH in buffer B, 2 pl of 0.5 M Nbs, solution (in 96% ethanol) was added, followed by the enzyme preparation, and the increase in A,,, was followed. The rate of reaction tended to decrease during the assay, probably as a result of Nbs, attack on the active site of the enzyme. Purified thioredoxin reductase was also assayed by its ability to reduce insulin [ 5 ] : the enzyme was added to a cocktail containing 0.2 mM NADPH, 0.5 mg/ml bovine insulin and 1 pM rabbit liver thioredoxin in buffer B, and the decrease in A,,, was followed.

Affinity Chromatography 0 1 1 2'5'ADP-Sephurosc Colutntis

Reticulocyte lysates were made 20pM in haemin and 50 pg/ml in creatine kinase before passage through 2'5'ADP- Sepharose columns. Gel-filtered lysates prepared as described above, were loaded directly, with no further additions. The 2'5'ADP-Sepharose was washed in buffer A. In general, the columns were loaded with 4-6 column volumes of lysate or gel-filtered lysate at a flow rate of about 10 ml h cm- cross-

section; the size of the fractions collected was about 10 "; of the load volume. Aliquots of each fraction were tested for Glc6P dehydrogenase activity and for [35S]methionine incorporation in a 10-niin incubation as described in the preceding paper. The. fractions which showed maximum incorporation were pooled (unless Glc6P dehydrogenase activity was detected in these fractions, in which case the material was discarded) and were frozen in liquid nitrogen in 0.5-ml aliquots. To isolate the material bound to the column, the column was first washed with 3- 5 column volumes of 0.1 M KCI in buffer A and then eluted with 1 mM NADPH, 0.1 M KCI in buffer A with the fraction size reduced to about 15:< of the bed volume. The fractions were routinely assayed for Glc6 P dehydrogenase activity and often for thioredoxin reductase (by the Nbs, reduction assay) and pooled as appropriate. This material will be called the NADPH eluate. After elution with NADPH, the column was washed extensively with 1 M potassium phosphate pH 7.5, and then re-equilibrated with buffer A before the next run.

Prcparat ion of' Rcticulocyte Glucosc.-h-phosphaie DPliydYogeimse

The NADPH eluate from a 2'5'ADP-Sepharose column that had been loaded with reticulocyte lysate was gel-filtered in 10mM Tris/HCl pH 7.5, 1 mM EDTA, and then chromato- graphed on a DEAE-Sepharose CL 6B column which had been equilibrated in the same buffer. Elution was with a linear gradient of 0-0.3 M NaCl in 10 mM Tris/HCl p H 7.5, 1 mM EDTA. The fractions with peak activity were pooled. Sodium dodecyl sulphate gel electrophoresis of this material suggested that purity was at least 75 %.

Pur$cation of Rabbit Liver Thioredoxin Reductase

Rabbit livers were homogenised in approximately 3 vol. of 0.25 M sucrose, 0.1 M KCI, 5 mM MgCI,, 14 mM 2-mercapto- ethanol, 20mM Tris/HCl pH 7.5, using a Waring blcnder for the first stage and finishing with a Dounce homogeniser. A postribosomal supernatant fraction was prepared by centrifugation for 2 h at 40000 rev./min in the Beckman 42.1 rotor. A 35 - 65 ammonium sulphate cut was made, desalted on a Sephadex G-50 column equilibrated with 10 mM TrisiHCl pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol and adsorbed to a DEAE-Sepharose CL 6B column equilibrated in the same buffer. The column WHS washed with 0.1 M NaCl in the same buffer to elute glutathione reductase and was then eluted with a linear gradient of 0.1 - 0.3 M NaCl in this buffer. The fractions with maximum activity in the Nbs, reduction assay were pooled and passed through a 2'5'ADP-Sepharose column equilibrated with 0.2 M NaCI, 10 mM Tris/HCl pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol. After extensive washing of the column with this buffer, thioredoxin reductase was eluted with the same buffer containing 1 mM NADPH. At this stage the material was more than 95 pure as judged by sodium dodecyl sulphate gel electrophoresis (see for example Fig. 2) but some preparations were purified further by glycerol gradient centrifugation.

Sodium Dodec.yl Sulphatel Polyatrylamide Gel Electrophoresis

Sodium dodecyl sulphate gel electrophoresis was performed using 15 "/, polyacrylamide slab gels prepared according to the formula in [6]. The gels were stained in Coomassie brilliant blue. Densitometry of the stained bands was carried out using a Transidyne 2955 scanning densitometer.

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01 0 5 10 15

Time [mini

Fig. I . Glucose 6-phosphutr dchydrogencrsr is yuri~ititutive1,y rrmovd 011

pussage through 2‘5’ADP-Srpharose. A 2’5‘ADP-Sepharose column (4-ml bed volume) was loaded with 17ml lysate, fractions of approximately 1.2 ml were collected, and Glc6P dehydrogenase assays conducted using 5- pl samples of each fraction and of the lysate load. The actual traces from the recording spectrophotometer are shown for fractions 10 and 12, the latter being the last fraction with haemoglobin concentration comparable to the load. The inset shows the results (plotted on the same scale as the main figure) of Glc6P dehydrogenase assay using a 5.~1 sample of the pooled column fractions, with the noise eliminated from the trace (- -). and the rate calculated for an activity equivalent to 17” of that in the fresh lysate load (--- -)

RESULTS

Preparation and Properties of‘ 2’S’A D P-Sepliarose- Trcaled Lysates

Lysates were made 20pM in haemin and 50pg/ml in creatine kinase before loading on to 2’5’ADP-Sepharose columns which had been equilibrated with the same low-salt buffer as was used for gel-filtration [I]. Fractions were pooled on the basis of a short-term protein synthesis assay using [35S]methionine as labelled amino acid and the pooled material was either used immediately or was stored as small aliquots in liquid nitrogen. Typically, 4- 5 column volumes of lysate were passed through the column, so the gel-filtration effect was negligible and the resulting lysates (which we will call 2’5’ADP lysates) contain all the low-molecular-weight components present in the parent lysate and all the macromolecular species except for those components adsorbed to the affinity column. The lysate load and the flow-through fractions were tested to see which enzymes had been adsorbed by the column and to quantify the efficiency of adsorption. The 2’5’ADP-Sepharose seemed to adsorb quantitatively glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase but did not bind isocitrate dehydrogenase to any significant extent, which is in agreement with published observations on the specificity of 2’5’ADP-Sepharose [2]. Fig. 1 shows typical results of Glc6P dehydrogenase assays of the load and flow-through fractions. It was not possible to detect activity in the effluent lysate and we estimate that at least 99.8 of the Glc6P dehydrogenase present in the lysate was bound to the column, the limit of the estimate being set by the sensitivity of the assay.

It proved impossible to make reliable quantitative assays of thioredoxin reductase activity in crude lysates and flow-

Fig. 2. Sodium dodecyl sulpliatr gel elertrop11orcsi.s of N A D P H e1uutr.v of 2’S‘ADP-Scpharuse columns. Tracks 1-3 and 6-9 were samples of NADPH eluates from 2’5’ADP-Sepharose columns which had been loaded with: normal reticulocyte lysate (tracks 1 - 3, three different preparations), Sephadex G-25 gel-filtered lysate (tracks 6 and 7, two different preparations), and rabbit liver postribosomal supernatant (tracks 8 and 9). Tracks 4 and 5 are two preparations of rabbit liver thioredoxin reductase prepared as described in Methods; these tracks have been overloaded to show the purity of the product. The track labelled M has marker proteins. ‘The arrows on the right side show the positions of thioredoxin reductase (T) and Glc6P dehydrogenase ((3). Analysis was on a 15 U< polyacrylamide slab gel which was stained with Coomassie brilliant blue, destained and photographed

through fractions, although qualitative tests suggested that this enzyme was not efficiently adsorbed by the column and was present in the effluent lysate. To examine this question in more detail, the proteins specifically adsorbed to the column were eluted with 1 mM NADPH and the eluate tested for various enzyme activities and analysed by sodium dodecyl sulphate gel electrophoresis. Not surprisingly, high Glc6P dehydrogenase activity was found in the NADPH eluate, but very little thioredoxin reductase activity (data not shown), nor could thioredoxin reductase be detected on gel electrophoresis (Fig. 2). The supposition that thioredoxin reductase was not efficiently adsorbed when crude lysates were passed through 2‘5‘ADPSepharose columns was confirmed by the discovery that efficient adsorption did occur if the lysate was gel-filtered (using Sephadex G-25) before passage through 2’5’ADP- Sepharose columns. The NADPH eluate from 2’5’ADP- Sepharose columns contained 5 - 10-fold more thioredoxin reductase activity (relative to Glc6P dehydrogenase activity) if gel-filtered lysate had been loaded on the column than if normal (not gel-filtered) lysate had been applied (data not shown). This difference was confirmed by analysis of the NADPH eluates by sodium dodecyl sulphate gel electrophoresis (Fig. 2) : densitometry of the stained bands indicated that the ratio of thioredoxin reductase to glucose-6-phosphate dehydrogenase was at least sixfold greater in eluates derived from gel-filtered lysates as opposed to normal lysates. These results show that when normal (not gel-filtered) lysates are passed through 2’5’ADP- Sepharose columns, at least SO-SO% of the thioredoxin reductase activity is retained in the flow-through and is not adsorbed to the column. Although these observations do not rigorously prove that the adsorption of thioredoxin reductase to the column is absolutely quantitative when gel-filtered lysates are applied, the properties of the effluent lysate which

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0 15 30 1 5 60 Time (min)

Fig. 3 . Kinetics o f r o t e i n syv?,nthcsi.s in 2’5’ADP /wurc,s. Incorporation assays using [‘4C]valine were carried out undcr standard conditions with the following additions: none (0 - +), 0.2 mM Glc6P (A -- A), 0.5 mM dithiothreitol (n-.--n), 0.2mM isocitrate (@-- -@), 30 pM NADPH (m - ~ ~- m), 7 pg/ml reticulocyte (ilc6P dehydrogenasc (A- - -A), and 0.2mMGlc6Pwith7 ~ilg/mlreticulocyteGlc6Pdehydrogenase( + - -+)

will be described later in this paper suggest that this enzyme is absent and that adsorption must have been complete.

The pcrformance of the 2’5’ADP-Sepharose columns was routinely monitored by assaying Glc6P dehydrogenase activity in the flow-through fractions, and only those preparations which showcd no detectable Glc6P dchydrogcnase were used for the studies of protein synthesis activity reported here. This proved to be an important safeguard against overloading the column: after about 10 column volumes of lysate had been passed through. a trickle of Glc6P dehydrogenase activity began to be detected. More serious was the fact that after sevcral runs the 2’5’ADP-Sepharose ceased to bind Glc6P dehydrogenase quantitatively, even though we followed the recommended washing procedures between each run, as described in Matcrials and Methods. Provided this precaution of monitoring thc performance of the column is taken, it can be concluded that when normal lysates arc passed through 2’5’ADP-Sepharose under the conditions used here, the effluent lysate contains all the low-molecular-weight metabolites of thc parent lysate and virtually all the isocitrate dehydrogenasc and thioredoxin reductase activities, but is totally devoid of Glc6P dehydrogenase, 6-phosphogluconate dehydrogenase and glutathionc reductase.

Pi.otf>iii Sjvitlwsis it7 2’5‘ADP Lysutes

The initial rate of protein synthesis in 2’5’ADP lysates was close to that of the parent lysate, but the rate fell precipitously after about 20 min of incubation (Fig. 3), just as in the case of gel-filtered lysates [ I ] . However, neither the addition of 0.2mM glucose 6-phosphate nor of any other sugar phosphate prevents the shut-off i n 2’5’ADP lysates, which is not altogether surprising since these lysates will contain the same pools of

sugar phosphates as the crude (not gel-filtered) lysate, as explained in the preceding section. On the other hand, either isocitrate or dithiothreitol were very effective activators of protein synthesis in these lysates and typically maintained protein synthesis at a linear rate for 60min or more (Fig. 3). Dose-response experiments showed that half-maximal response was obtained at about 20 pM dithiothreitol or 40 pM isocitrate (data not shown). Addition of NADPH at low concentrations stimulated protein synthesis quite efficiently (Fig. 3), although complete reactivation was never observed even when higher concentrations werc used; in fact 30- 40 pM was sufficient to elicit maximum response. Nevertheless it is intcresting that NADPH was far more efficient in these lysates than in gel-filtered lysates.

Activation of protein synthesis could also be achieved by the addition of the crude NADPH eluate from the 2’5’ADP- Sepharose column, or partially purified reticulocyte Glc6P dehydrogenase (Fig. 3), or commercial Glc6P dehydrogenase from yeast. In some preparations of 2‘5’ADP lysates Glc6P dehydrogenase alone was sufficient to maintain a high rate of protein synthesis for 60 min, but the more usual result was that maximum response required the addition of both Glc6P and Glc6P dehydrogenase as is the case in Fig. 3. This variability between different preparations presumably rcflects differences in the endogenous level ofGlc6P in different batches oflysate. It should be born in milid that a 2’5’ADP lysate supplemented with Glc6P dehydrogenase is not exactly the same (untreated) lysate : 6-phosphogluconate dehydrogenase is absent and therefore glucose 6-phosphate and other sugar phosphates are unable to recycle through the pentose phosphate pathway. The endogenous pool of sugar phosphates in lysates may be inadequate to satisfy the requirements of 2’5’ADP lysates even though these levels may be sufficient to sustain protein synthesis in the parent lysate where recycling can occur.

The activation of protein synthesis by the addition of reticulocyte Glc6P dehydrogenase to a 2’5’ADP lysate supplemented with 0.2 mM glucose 6-phosphate was half-maximal at about 0.15 pg/ml final concentration of Glc6P dehydrogenase and maximal at about 0.75 pgjml. Since we estimated that lysates typically contain about 10 pg/ml Glc6P dchy- drogenase, it is clear that this enzyme is present in normal (untreated) lysates at levels far in excess of the amount needed to sustain protein synthesis activity. This is consistent with the finding that a mixture of as little as 5 7” (by volume) of normal lysate and 95 2’S’ADP lysate maintained the maximum rate of protein synthesis for 60min (data not shown).

N o loss of activity occurred when 2’5’ADP lysates werc frozen in liquid nitrogen and stored inliquid nitrogen, but these lysates did lose activity much more rapidly than the parent lysate on ‘ageing’ at 0 ’C. This loss of activity was manifest mainly as a reduction in the rate of protein synthesis, but there was also a tendency for the shut-off to occur earlicr after ‘ageing’. The addition of the critical compounds discussed in this section still prevented the shut-off and maintained linear rates of protein synthesis, but none of them overcame the decrease in the rate of synthesis, the cause of which remains unknown. In a few, not entirely typical, ‘aged’ 2’5’ADP lysates, the ability to respond to NADPH, isocitrate or GlchP dehydrogenase was partially lost, although the lysate still showed full response to dithiothreitol. In these cases activation by NADPH-generating systems was enhanced to the same level a s was observed using dithiothreitol if thioredoxin reductase was added, so it seems likely that in such lysates ageing at 0 C had resulted in the inactivation of thioredoxin reductase.

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Preparation aiid Properties qf‘ Gel- FiltPred 2‘SA D P Lysutrs

In order to make a more complete definition of the components necessary to prevent the shut-off of protein synthesis, i t was necessary to examine lysates which had been both gel-filtered (to remove low-molecular-weight metabolites) and passed through a 2’5‘ADP-Sepharose column to remove critical NADP/NADPH-utilising enzymes. Our first attempts towards this objective used lysates which were first put through a 2’5’ADP-Sepharose column and subsequently gel-filtered. According to the results described earlier in this paper, such lysates should be expected to retain almost the full complement of thioredoxin reductase present in normal (untreated) lysates and they would differ from 2’5’ADP lysates only in their lack of low-molecular-weight metabolites. These lysates exhibited rather low rates of protein synthesis: in this respect they were very similar to the typical ‘aged’ 2’5’ADP lysates described in the previous section and we imagine that the poor activity was a consequence of the ‘ageing’ during the time required for the gel- filtration step. Not surprisingly, protein synthesis in such lysates shuts off after about 10 min of incubation. To prevent this shut-off and to maintain linear rates of protein synthesis it was necessary to add both a sugar phosphate (glucose 6- phosphate, or 2-deoxyglucose 6-phosphate or fructose 1,6- bisphosphate) and a reducing system, which could be either dithiothreitol or an NADPH-generating system (either isocitrate, or Glc6P dehydrogenase if Glc6P had been used as the sugar phosphate, or even NADPH itself). An example of one of the more active preparations we obtained can be seen in Fig. 1 of the following paper. The addition of thioredoxin reductase did not enhance or potentiate the activation of protein synthesis by NADPH-generating systems. Although these combinations of agents prevented the shut-off and sustained linear rates of protein synthesis for 60 min they did not enhance the low initial rate of incorporation, a further point of similarity between these lysates and the ‘aged’ 2’5’ADP lysates described in the preceding section.

Since the low rate of synthesis in these lysates was un- satisfactory, we examined lysates which had first been gel- filtered (Sephadex (3-25) and then passed through a 2’5’ADP- Sepharose column. These lysates were much more active and were used in all subsequent work: they will be called G 2 5 / 2’5’ADP lysates. The initial rate of protein synthesis was at least 50 %, and more typically about 80 :/<, of the rate observed in the parent gel-filtered lysate. These preparations lost no activity on freezing and storage in liquid nitrogen but on ageing at 0 °C the decline in activity occurred faster even than in the case of 2’5’ADP lysates. I t was therefore important to prepare such lysates as quickly as possible and to free7e down aliquots as soon as the flow-through fractions from the 2’5’ADP- Sepharose column had been assayed for Glc6P dehydrogenase activity (to monitor the performance of the column) and a short-term protein synthesis assay using [3sS]methionine had been carried out to determine which fractions should be pooled.

The results described earlier in this paper (Fig. 2) show that when gel-filtered lysates are passed through 2’5’ADP- Sepharose columns the adsorption of thioredoxin reductase to the column is much more efficient than when normal (untreated) lysates are loaded on the column. Evidently, some low- molecular-weight metabolites present in the lysate must prevent efficient binding of this enzyme to the affinity column, but experiments to identify the critical compound(s) have not been carried out. The important point is that G-25/2’5’ADP lysatcs not only lack all the low-molecular-weight metabolitcs, Glc6P dehydrogenase, 6-phosphogluconate dehydrogenase and

glutathione reductase present in normal lysates, but also contain very little, if any, thioredoxin reductase. In contrast, they retain almost the full complement of isocitrate dehydrogenase.

Proteirt Syrzrllesis i t i G-25/2’S‘ADP Lysates

The initial rate of protein synthesis in these lysates was up to of the rate in the parent (3-25 lysate, but this rate

ined for only about 10min before inhibition set in (Fig. 4). The shut-off in these lysates therefore occurred earlier than in either G-25 lysates or 2’5’ADP lysates. I t was necessary to add both sugar phosphates and a reducing agent (e.g. 0.2 mM Glc6P plus 0.5 mM dithiothreitol) to prevent this shut- off and to maintain the high initial rate of synthesis for 60 min (Fig. 4). In the absence of dithiothreitol, neither glucose 6- phosphate nor any other sugar phosphate stimulated protein synthesis significantly or delayed the onset of inhibition (Fig. 4 and Table 1) . The response of these lysates to dithiothreitol in the absence of sugar phosphates was somewhat variable between the partial activation shown in Fig.4 and a very modest stimulation above controls lacking dithiothreitol, as is the case in the experiment shown in Fig.2 of the following paper [7]. The important point is that in all such lysates only a combination of sugar phosphate and dithiothreitol allowed full activation.

The requirement for glucose 6-phosphate (in the presence of dithiothreitol) could also be satisfied by 2-deoxyglucose 6- phosphate or fructose 1,6-bisphosphate (Table I ) . On the other hand 6-phosphogluconic acid was at best a very poor substitute for Glc6P, as in the experiment shown in Fig. Y of the following paper [7], and in most experiments it did not stimulate protein synthesis over that which could be obtained with dithiothrcitol alone in the absence of sugar phosphates (Table l) , even though the parent (3-25 lysate was fully activated by 6-phosphogluconate. The dose-response of G-25/2’5’ADP lysates to sugar phosphates and to dithiothreitol was assayed by the same method as in Fig. 7 of the preceding paper [l]. The concentrations required for half-maximal stimulation are sum- marised in Table2 and can be seen to be similar to those for G-25 lysatcs [I]. The most significant difference is that gel- filtered lysatcs seemed to require higher concentrations of 2-dc- oxyglucose 6-phosphate than did G-2512’5’ADP lysates. One possible explanation for this difference is that in gel-filtered lysates 2-deoxyglucose 6-phosphate has to act both as a sugar phosphate and as an NADPH generator, whereas it is only required to serve the role of a sugar phosphate in thesc assays of G-25/2’5’ADP lysates, since the reducing agent is supplied as dithiothreitol.

In experiments in which dithiothreitol was replaced by an NADPH-generating system (e.g. isocitrate, or Glc6P dehydrogenase if Glc6P was added as the sugar phosphate) or NADPH itself, no stimulation of protein synthesis was observed (Fig. 4). On the other hand, full stimulation was obtained by the addition of Glc6P and the crude NADPH eluate from a 2’5’ADP-Sepharose column which had been loaded with rabbit liver postribosomal supernatant (Fig. 4). Although this eluate is known to contain Glc6P dehydrogenase and NADPH, control experiments showed that these are not sufficient to activate protein synthesis (Fig. 4) and the eluate must have contributed some other essential component. I t seemed likely that this additional component was thioredoxin reductase and experiments to be discussed in more detail in the following section showed that the addition of purified rabbit liver thioredoxin reductase together with G M P , Glc6P dehydrogenase and NADP promoted maximal activation of protein

308

Time lmin)

Fig. 4. Kinetics ofprotein synthesis in G-25/2‘5’ADP lysates. Incorporation of [14C]valine in a G-25/2’5’ADP lysate was assayed under standard conditions with the additions specified below at the following final concentrations: 0.2 mM Glc6P, 0.2 mM isocitrate, 0.5 mM dithiothreitol, 50 pM NADPH, 5 pM E. coli thioredoxiii, 0.25 pM E. coli thioredoxin reductase and 7 pg/ml reticulocyte Glc6P dehydrogenase. (A) N o additions (0-): Glc6P (O-); Glc6P and isocitrate (A- - A); dithiothreitol ( 0 4 ) ; Glc6P and dithiothreitol (+ -+). (B) Glc6P, thioredoxin and thioredoxin reductase (A- --A); isocitrate, thioredoxin and thioredoxin reductase (*--+I; G M P , isocitratc, thioredoxin and thioi-edoxin reductase (A- -A); Glc6P, NADPH, and Glc6P dehydrogenase (0- 0); Glc6P plus NADPH eluate from a 2’S’ADP-Sepharose column loadcd with rabbit liver postribosomal supernatant (.----a). In the assay containing the crude NADP€I eluate, the final concentration of NADPH was 50 IM, and the Glc6P dehydrogenase added was cquivalent in activity to a final concentration of about 5 pg/ml reticulocyte Glc6P dehydrogenase. A and B are the results of a single experiment, displayed on two panels for clarity

Table 1. Sugar phosphate requiremenis ,/#r activuiiun o/proiein syizthesh Incorporation of [‘4C]valine in a G-25/2’5’ADP lysate supplemented with sugar phosphates was assayed under standard conditions and a 60-min incubation time with the following additions, where specified: dithiothreitol(O.5 mM), isocitrate (0.2 mM), E. coli thioredoxin (3 pM), E. col i thioredoxin reductase (0.15 pM). After a 10-min incubation, incorporation in the assay containing 0.1 mM Glc6P and 0.5 mM dithiothreitol was 181 3 counts/min. The last column shows the incorporation in the parent G-25 lysate assayed under the same conditions (60-min incubation) with the same final sugar phosphate concentrations. n.t. = not tested

Additions (Concn) Incorpordtion of [‘4t]valine by

G-2512’5 ADP lysate parent

-reducing agent + dithiothreitol + isocitrate, thioredoxin,

~ __- ~ _ _ _ _ _ _ - _ _ - ~- ~

~~ _ _ ~ - -~ ~ G-25 lysate

thioredoxin reductdse

coLints/mui __ - - -~ __ ~- ~-

(IM)

None 2502 5 626 6 777 4696

Glc6P Glc6P Fru(l,6)P2 Fru(l,6)P2 2dGlc6P 2dGlc6P 6-P-Gluconate Ribulose-5-P

2 843 n.t.

2741 1l.t.

2 605 n.t.

2289 2679

10685 6905

11 209 6 309 9 947 6638 5 229

10814

11 535 7 561

12 634 6751

11 394 7 999 5 782

11815

10009 n.t.

7 809 n.t. n.t. n.t.

10401 n.t.

synthesis (Table 4). Dose-response assays using a G-25/ 2’5’ADP lysate supplemented with 0.2 mM Glc6P to fulfill the sugar phosphate requirement and 0.2 mM isocitrate to generate NADPH, showed that half-maximal response was obtained with a final concentration of 0.5nM rabbit liver thioredoxin reductase (Table 2) and maximal activation with approximately 2 nM (assuming an M , of 110000 for native thioredoxin reductase). The addition of rabbit liver thioredoxin was not necessary for activation of protein synthesis by thioredoxin reductase, nor did it seem to potentiate activation (data not shown). This is not altogether surprising, since a

G-25/2’5’ADP lysate should retain the full complement of thioredoxin present in the crude (untreated) lysate.

In further experiments, purified E. coli thioredoxin reductase was used, but in this case the presence of E. coli thioredoxin was an absolute pre-requisite for stimulation of protein synthesis, in addition to the requirements for sugar phosphates and a source of NADPH (Table 3). The nccessity for E. coli thioredoxin despite the fact that G-25/2’5’ADP lysates should contain (mammalian) thioredoxin, is under- standable in the light of reports that E. coli thioredoxin reductase is specific for the homologous (bacterial) thioredoxin

Table 2. Concentrutions of uctiraririg c~ornpounrf,~ rcquirrd fo r lin~f-nza.uiti7ul s~imuluti~in of’proiein synrhesis it? G-2512’5’A DP lysu~rs Dose-response experiments were carried out using a 60-min incubation period and the concentrations required for half-maximal activation determined. Dose-response assays for sugar phosphates were carried out in the presence of 0.5 mM dithiothreitol, for dithiothreitol in the presence of 0.2 mM Glc6P and for all tests of thioredoxin reductax and thioredoxin i n the presence of 0.2mM Glc6P and 0.2 mM isocitrate. All assays with E. coli thioredoxin and thioredoxin reductase contained the two components in a 20: 1 molar ratio

Addition Conce n t w t ion required for half-maximal activation

CL M Glucose 6-phosphate 15-20 2-Deoxyglucose 6-phosphate 40 Fructose I ,6-bisphosphate 125 Dithiothreitol 20 Rabbit liver thioredoxin reductase 0.0005 E. coli thioredoxin reductase 0.008 E. coli thioredoxin 0.16

Table 3. The requirementfi)r both E. coli rhiorehxiri mid b,. c d i iliiorcclo.uiii reductuse ,for ucrivutiun o f protein syiithesis lncorporation of [‘4C]valine in a G-25/2’5’ADP lysate was assayed under standard conditions in a 60-min incubation with the additions indicated below at the following final concentrations: Glc6P(0.2 mM), dithiothi-eitol (0.5 mM), isocitrate (0.2 mM), B. coli thioredoxin ( 5 pM), E. coli thlorcd- oxin reductase (0.25 pM). After a 10-min incubation, incorporation in the sample containing Glc6P and dithiothreitol was 1100 counts/min

Additions Incorporation of [I 4C]valine

-Glc6P +Glc6P

None Dithiothreitol Isocitrate Thioredoxin Thioredoxin, isocitrate Thioredoxin reductase Thioredoxin reductase, isocitrate Thioredoxin, thioredoxin reductase Thioredoxin, thioredoxin reductase,

isocitrate

countsimin

1511 3278 1676 1317 1399 1486 1459 1461

-

3407

_ _ 1544 6805 1611 1572 1551 1572 1506 1746

7221

and cannot reduce mammalian thioredoxin [4]. These results therefore support the view that what is required for activation of protein synthesis is a complete functional thioredoxin/thioredoxin reductase system, and that the stimulation observed in these experiments was not due to contaminants in the enzyme preparations.

The purified E. coli thioredoxin and thioredoxin reductase were added to these assays in an arbitrarily chosen molar ratio of 20: 1 ; half-maximal stimulation (in the presence of 0.2 mM Glc6P and 0.2 mM isocitrate) required final concentrations of 8 nM thioredoxin reductase and 0.16 pM thioredoxin (Table 2) and maximal activation 70 nM and 1.4pM respectively. Although no experiments have been carried out to determine

Table 4. Protein syiithesis is uctivuied by the fizioredosin sysirr77 hul t1ot h)’ &tathione Incorporation of [‘4C]valine in a G-25/2’5’ADP lysate was assayed with the additions indicated under standard conditions and a 60-min incubation time. After 10min of incubation incorporation in the most active sample (last line of table) was 1298 counts/min

Additions (find concentration) Incor-

0 2 m M Glc6P, 1 mM GSH, 0 4 pg/ml 10 pg/ml Glc6P 10 pg/ml GSSG thioredoxin dehydrogenase, reductase reductase IOpM NADP

por‘ition ~ _ _ _ ____ -~ _ _ _ _ - -

~

+ + + +

counh/min

- 183‘) - 2301 - 2839 + 5796 + 5966

which of the two components was limiting in these assays, it is clear that the thioredoxin system is considerably more effective at lower concentrations than dithiothreitol. When E. coli thioredoxin and thioredoxin reductase were used in place of dithiothreitol, maximum activation of protein synthesis still required the presence of sugar phosphates (Fig. 4 and Table 1) and the specificity with respect to different sugar phosphates is the same as when dithiothreitol is used (Tablel). Although detailed studies of the dose-response of the system to sugar phosphates in the presence of E. coli thioredoxin and thioredoxin reductase have not been carried out, the results in Table 1 demonstrate that the concentrations required for half-maximal activation are fairly close to the levels needed when dithiothreitol is used, but may bc marginally lower.

Glutatlziow Does iw t Activate Prolein Synthesis

The results described in the previous section show that the rnaintenancc of high rates of protein synthesis in G-25/ 2’5’ADP lysates requires the presence of sugar phosphates and a suitable reducing agent: either dithiothreitol or an NADPH-generating system together with an active thioredoxin/thioredoxin reductase system. It has bcen found that the role of thioredoxinlthioredoxin reductase in de- oxyribonucleotide synthesis in E. coli can be substituted by glutathione in conjunction with glutathione reductase and glutaredoxin, a small protein which has many properties similar to thioredoxin except that i t is reduced by glutathione and not by thioredoxin reductase [8,9]. Since reticulocyte lysates contain high concentrations (1.5 - 2.0 mM) of glutathione, it is possible that glutathione is as important a reducing agent in lysates as is the thioredoxin system. Glutaredoxin has been detected in mammalian cells [lo] and we have isolated a small protein from reticulocyte lysates which catalyses the glutathione-driven reduction of hydroxyethyldisulphide and cystamine, and therefore appears to be Cunctionally identical to glutnredoxin [8,9]. Since this protein is of similar size to thioredoxin [lo], it should be retained in a G-25/2’5’ADP lysate, so that such lysates would need to be supplemented with an NADPH-generating system (e.g. Glc6P plus Glc6P dehydrogenase), glutathione and glutathione reductase, in order to reconstitute the complete glutathione/glutaredoxin system. Table4 shows that when these additions were made there was

310

Table 5 . Ti7e c@,ct yf GTP ai idr ,AMP oi i j ) rof~& .syiithesis in G-ZSj2S’A D P lysares Incorporation of [‘“Clvaline in a G-25/2’5’ADP lysate was assayed with the additions indicated (linal concentrations) under standard conditions and a 60-min incubation time. After 10 rnin of incubation, incorporation in the assay containing 0.2mM Glc6P and 0.5mM dithiothreitol was 1594 counts/min. n.t. = not tested

Additions Incorporation oi [ I 4C]vni~nc _ - - - - ~.

Glc6P GTP MgCI2 CAMP -dithio- +dithio-

(0 5 mM) threitol threitol

mM counts/min

- - - _ 1752 6207 - - 2104 9 041 0 2 -

- 2 2 4405 10358 0 2 2 2 5590 n t - - - 4 41 34 9283

_ 4 4617 11 t 0 2 -

__ - ~- -~ - -~

_

_

no activation of protein synthesis, although the addition of mammalian thioredoxin reductase and the NADPH- generating system promoted full activation. Even when the concentrations of glutathione and glutathione reductase were varied over a wide range, no stimulation of protein synthesis occurred and the shut-off occurred at the same time as in controls lacking any of these components (data not shown). We conclude that the thioredoxin/thioredoxin reductase system is absolutely necessary to maintain full rates of protein synthesis (in the absence of dithiothreitol) and is apparently sufficient for this purpose; there is no evidence for a role of glutathione in this process, not even as an auxiliary component.

Activation of Proteiri Synthesis by GTP arid c A M P

Protein synthesis in gel-filtered lysates is stimulated by GTP and by high concentrations of cAMP [l, 2 1 - 141, two compounds which do not conform to the rule that activators are either sugar phosphates or potential reducing agents (or both). It was therefore of interest to test whether these compounds affected protein synthesis in 2’5’ADP lysates and G-251 2’5’ADP lysates, particularly as in the latter type of lysate it should be possible to distinguish whether GTP and cAMP were more effective as substitutes for dithiothreitol or for sugar phosphates. Both types of lysate were stimulated by GTP or by CAMP, but to a lesser extent than was the case with gel-filtered lysates: 2 mM GTP (with 2 mM MgCI,) or 4 mM cAMP rarely stimulated protein synthesis to more than half the maximum activity attainable. Table 5 shows typical results obtained using a G-25/2’5’ADP lysate. Whilst GTP or cAMP were somewhat stimulatory when added alone, there was strong synergism between these compounds and dithiothreitol and to a lesser extent with GlchP. I t was only in combination with dithiothreitol that either GTP or cAMP were able to effect full activation (Table 5). One interpretation that might be placed on this result is that GTP and cAMP more closely mimic the effects of Glc6P than the effects of dithiothreitol, but in view of the complexities of the system such an interpretation may be too simplistic.

DISCUSSION

These experiments use affinity chromatography to remove selectively alimited number of enzymes from the lysate without inactivating or removing any of the numerous components of the basic protein synthesis machinery. This has allowed us to test the effects of specific enzymes, added either singly or in combinations, and has made i t possible to answer questions which were otherwise inaccessible to study. Up to now, the traditional biochemical approach of fractionation and purifi- cation of the relevant proteins, followed by reconstruction of a defined system, has contributed very little to understanding the control of eukaryotic translation. Such reconstituted systems have so far been of low activity and have failed to display the control mechanisms known to operate in intact cells and in crude extracts. We havc obviously been lucky that an appropriate affinity medium is available that is highly selective in binding precisely those enzymes which we wished to remove. It is not clear whether this approach has more general applica- bility to the study of protein biosynthesis, but the success of our experiments suggests that further applications may be worth pursuing.

The results presented in this paper fully reveal the dual requirements for the maintenance of high rates of protein synthesis that was hinted at by the behaviour of gel-filtered lysates [l]. These requirements are: (a) the presence ofa suitable sugar phosphate, and (b) a suitable reducing system, which may be either dithiothreitol or an NADPH-generating system plus a functional thioredoxin/thioredoxin reductase system. The par- ticular features of the reducing system suggest the need to reduce disulphide bonds; experiments designed to examine this point are presented in the following paper [7]. Although it is operationally useful that dithiothreitol can fulfill the requirement for reducing power, it is of no physiological significance. There seems little doubt that it is the thioredoxin/thioredoxin reductase system that is operative in the cell. I t is perhaps surprising that glutathione appears to play no role in the control of protein synthesis. Despite the high concentrations of GSH and GSSG reductase in reticulocytes, the evidence against their participation in maintaining high protein synthesis activity is quite clear cut.

The results of this paper show unambiguously that the sugar phosphate requirement is quite independent of and is unrelated to the generation of NADPH. Since full stimulation of protein synthesis requires the presence of a sugar phosphate in addition to a suitable reducing system, it is rather meaningless to ask which is the more essential, but inasmuch as G-25/2‘5‘ADP lysates consistently show no response to sugar phosphates alone but are partially activated by a reducing system in the absence of sugar phosphates, the reducing agent appears to be the more critical of the two.

The sugar phosphate requirement can be satisficd by GlchP, 2dGlc6P or Fru(l,6)P2 (or compounds which can be metabolised to these sugar phosphates) but not by 6-phosphogluconic acid. The ability of 6-phosphogluconate to stimulate protein synthesis in most G-25 gel-filtered lysates and some G-50 lysates [I] is therefore most likely to be dependent on its conversion to Glc6P via the pentose phosphate pathway, a conversion which cannot take place in 2’5’ADP lysates because 6-phosphogluconate dehydrogenase is missing. The fact that 2dGlc6P stimulates protein synthesis in G-25/2’5’ADP lysates containing dithiothreitol is highly significant since 2dGlc6P would be expected to be metabolically inert in such lysates: its structure precludes conversion to fructose derivatives, whilst the absence of Glc6P dehydrogenase eliminates the possibility

31 I

of conversion to 2-deoxy-6-phosphogluconate. This argues that the sugar phosphates d o not have to be metabolised to fulfill their role as activators of protein synthesis and con- sequently they would appear to be acting directly as cofactors in some way.

In the following paper, the reasons for the dual requirement and the roles of the sugar phosphates and the reducing system are examined in detail.

This work was supported by a grant from the Medical Research Council. T. H. was the holder ofthc Alan Johnbton. Lawrence and Moscleq Research Fellowship of the Royal Society. We thank Dr A. Holmgren for his invaluable advice and for the gift of purified bacterial thioredoxin and thioredoxin reductase.

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3. Pigiet, V. P. & Conley, R. R. (1977) J . Biol. Chem. 252, 6367-6372. 4. Holmgren, A. (1977) J . Bid. Chem. 252, 4600-4606. 5 . Holmgren, A. (1979) J . Bid. C h m . 254, 9113-9119. 6. Anderson, C. W., Baum, P. R. BGesteland, R. F. (1973)J. Virol. 12,

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T. Hunt, P. Herberl, E. A. Campbell, C. Delikadis, and R. _I. Jackson, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, Great Britain, CB2 lQW

the use of affinity chromatography on 2′5′ adp-sepharose reveals a requirement for nadph,...

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