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Home , Phosphoglycerate kinase, Phosphoribulokinase

Eur. J. Biochem. 173,437-443 (1988) 0FEBS 1988

A functional five- complex of involved in the

Brigitte GONTERO ’, Maria Luz CARDENAS’ and Jacques RICARD’ Centre de Biochimie et de Biologie Moleculaire du Centre National de la Recherche Scientifique, Marseille Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Santiago

(Received October 15, 1987/January 4, 1988) - EJB 87 11 55

A complex of five different : ribose- , , ribulose-bisphosphate carboxylase/oxygenase, phosphoglycerate and glyceraldehyde-phosphate dehydrogenase, has been purified from spinach chloroplasts. These enzymes catalyse five consecutive reactions of the Calvin cycle, of which the two reactions catalysed by phosphoribulokinase and ribulose-bisphosphate carboxylase/oxygenase are unique to this cycle. The five-enzyme complex has been purified by successive chromatographies on DEAE-Trisacryl, Sephadex G-200 and hydroxyapatite. The homogeneity of the complex has been tested by analytical centrifugation and electrophoresis. Depending on the technique used to estimate its molecular mass, the value obtained varies between 520 kDa and 536 kDa. In addition to the five enzymes mentioned above, the complex contains a 65-kDa polypeptide. The quaternary structure of these enzymes in the complex appears to be different from what has been described for the individual enzymes in the ‘noncomplexed state’. The five-enzyme complex is functional as glyceraldehyde 3-phosphate is formed from ribose 5-phosphate. Preliminary experiments suggest that channelling of reaction intermediates occurs within the complex.

It has been shown in recent years that, besides tightly MATERIALS AND METHODS associated enzymes forming stable complexes, there exist Mater ials loose or transitory associations of enzymes forming unstable complexes [l, 21. These loose associations have been described Spinach plants (Spinacea oleracea var. Giant d’Hiver) both for of the cytosol[3] and for those of mitochon- were grown in a research garden in Aix-Marseille I1 Univer- dria [4]. Stable or unstable enzyme complexes may represent sity. Ribulose Sphosphate, ribulose 1,5-bisphosphate and a functional advantage for the living cell, as compart- yeast ribose-phosphate isomerase were from Sigma, bovine mentalization may allow channelling of an intermediate from serum albumin from Calbiochem and the other reagents from one to another within the same enzyme complex Boehringer. Trisacryl was obtained from IBF Biotechnics [5, 61. If these associations are loose they may obviously be (France), Sephadex G-200 and Sephacryl S-300 from disrupted in the course of the purification procedure, thus Pharmacia. Deionized water, further purified with a Millipore making it difficult to identify the multienzyme complexes. super Q system, was used throughout. Thus only recently has a large multienzyme complex, the so- called ‘Krebs cycle metabolon’ [4], been shown to occur in Purification the multienzyme complex mitochondria. No similar complex has ever been reported in of the case of enzymes belonging to the Calvin cycle, although Intact spinach chloroplasts were isolated by the method some recent reports suggest the possibility of an association of Jensen and Bassham [9]. They were suspended in a buffer between certain enzymes of this cycle [7, 81. (15 mM Tris/HCl, 4 mM EDTA, 10 mM , pH 7.9) We report in this paper that five enzymes, ribose-phos- called buffer A, which brings about lysis of these organelles. phate isomerase, phosphoribulokinase, ribulose-bisphos- The whole extract was then centrifuged for 60min at phate carboxylase/oxygenase, , 48000 x g to remove membranes. The complex was isolated glyceraldehyde-3-phosphate dehydrogenase, which catalyse and purified from the supernatant by successive separations five consecutive reactions of the Calvin cycle, exist as a com- on DEAE-Trisacryl, Sephadex G-200 and hydroxyapatite. plex that may be isolated and purified, and which is The supernatant was applied first on a DEAE- functionally active. We also discuss some possible biological Trisacryl column (2.5 x 13 cm) equilibrated in buffer A and implications of the existence of this complex. washed with buffer A containing 50 mM NaC1. Elution of the column was performed with a gradient of 50 - 300 mM NaCl in buffer A. Fractions containing phosphoribulokinase ac- Correspondence to J. Ricard, Centre de Biochimie et de Biologie tivity were pooled, concentrated and submitted to molecular MolCculaire, BP 71, F-13402 Marseille Cedex 9, France sieve chromatography on a Sephadex G-200 (column Enzymes. Ribose-phosphate isomerase (EC 5.3.1.6); phospho- 2.6 x 95 cm) using another buffer, B, having the following (EC 2.7.1.19); ribulose-bisphosphate carboxylase/ composition: 30 mM Tris/HCl, 1 mM EDTA, 100 mM NaCl, oxygenase (EC 4.1.1.39); phosphoglycerate kinase (EC 2.7.2.3); 10 mM cysteine, pH 7.9. The fractions located in the void glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.13). volume contained ribose-phosphate isomerase, phosphori- 438

81 2 3 0. e EX6 0. 6 -11% .--I

<4 \E 3 3 0. 4

2 0. 2

0 0 100 150 200 250 300 350 Elution volume (ml)

B 0. 25 , 1

0. 20

0. 15 r( E 1 2 0. 10

0. 05

0 0 200 400 600 Elution volume Imll Fig. 1. Cvpurificution ofu high-moleculur-mussphosphoribulokinuseand other enzymes,from the Calvin cycle. Elution profiles of Sephadex G-200 and hydroxyapatite. The different enzyme activities were monitored as described in Materials and Methods. Activated phosphoribulokinase corresponds to the enzyme which has been incubated with 50mM for 1 h at 30°C before being assayed. (0-0) Phosphoribulokiuase, (0---0) activated phosphoribulokinase, (0-0) ribose-phosphate isomerase, ( A-A) glyceraldehyde-3-phos- phate dehydrogenase, (0-0) ribulose-bisphosphate carboxylase/oxygenase. (A) Gel filtration on Sephadex G-200. The arrows indicatc the predicted positions for elution of the isolated enzymes as follows: (1) ribulose-bisphosphate carboxylase/oxygenase, (2) glyceraldehyde-3- phosphate dehydrogenase, (3) phosphoribulokinase, (4) ribose-phosphate isomerase, (5) phosphoglycerate kinase. Column flow rate was 8 ml/h. The activity ratios, calculated in relation to activated phosphoribulokinase activity, can be considered constant within the experimental error value across the peak eluted in the void volume. Thus, the activity ratios for ribose phosphate isomerase, ribulose-bisphosphate carboxylaseloxygenase and glyceraldehyde-3-phosphatedehydrogenase were respectively 0.1 13, 0.064, 0.1 1 at 125.7 ml; 0.08, 0.055,0.1 3 at 151.30 ml; 0.048, 0.042, 0.096 at 164 ml; 0.062, 0.043, 0.091 at 176.80 ml; 0.098, 0.06, 0.085 at 189.50 ml. (B) Chromatography on hydroxyapatite. Column flow rate was 30 ml/h. The line (-) indicates the potassium phosphate concentration. The inset shows the activity ratios calculated in relation to phosphoribulokinase activity for ribose-phosphate isomerase (0-0) and glyceraldehyde-3-phosphate dehydrogenase (A-A) across peaks A and B bulokinase, ribulose-bisphosphate carboxylase/oxygenase, that described in [lo]. These activities were coupled to NADH phosphoglycerate kinase and glyceraldehyde-phosphate de- oxidation via and . hydrogenase activities. These fractions were pooled and dia- When measuring isomerase activity, the assay cuvette lysed against a third buffer, C, made up with 50 mM potass- contained 50 mM glycylglycine buffer pH 7.7,lO mM MgC!2, ium phosphate pH 7.2 and 10 mM cysteine. The resulting 0.5mM EDTA, 2.75 units lactate dehydrogenase, 1 unit extract was then loaded on a hydroxyapatite column pyruvate kinase, 1 mM phosphoenolpyruvate, 5 mM dithio- (2.5 x 14 cm). After washing with buffer C, the column was threitol, 0.15 mM NADH, 1 mM ribose 5-phosphate and eluted with a linear gradient of 50 - 200 mM potassium phos- 0.5 mM ATP. The same mixture, supplemented with ribose- phate. The fractions containing phosphoribulokinase activity phosphate isomerase from the yeast Torula, was used for were collected. measuring the kinase activity. Glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase and ribulose-bis- phosphate carboxylase/oxygenase activities were determined Enzyme assays as described in [ll - 131. The complex activity was measured Phosphoribulokinase and ribose-phosphate isomerase ac- using the ribulose-bisphosphate carboxylase/oxygenase assay, tivities were determined at 30°C by a procedure derived from but without the addition of exogenous phosphoribulokinase, 439

Table 1. Content of enzyme uctivities in peaks A and B of hydroxy- uputite The enzyme activities were assayed as described in Materials and Methods. The total content was 712 pg and 541 pg in peak A and B, respectively

Enzyme Peak A Peak B

units

Ribulose-bisphosphate carboxylase/ oxygenase 8.5 6.7 Phosphoribulokinase 6 5" Fig. 2. Gradient polyacrylamide gel slab: 4-30%. From left to right: Ribose-phosphate isomerase 3.7 3" track 1, 10 pg thyroglobulin; track 2, 10 pg complex A eluted from Glyceraldehyde-3-phosphate hydroxyapatite chromatography; track 3, 10 pg complex B eluted deh ydrogenase 0.3 1.4 from hydroxyapatite chromatography; track 4, mixturc of 10 pg Phosphoglycerate kinase 0.09 0.15 catalase, LO pg thyroglobulin, 10 pg ferritin; track 5, 10 pg RNA ; track 6, 10 pg ferritin; track 7, 10 pg catalase. Electro- a Assays were also done arter preincubation with 50 mM dithio- phoresis was done at 70 V for about 20 min, until the protein moved thrcitol for 1 h. The activities of both enzymes are enhanced from into the gel, then for 16 hat 150 V. Electrophoresis buffer was 90 mM 5 units to 17 units, and from 3 units to 7.5 units. Tris, 80 mM boric acid, 2.5 mM NazEDTA pH 8.4

ribose-phosphate isomerase, glyceraldehyde-phosphate de- phosphoribulokinase activities in the void volume of the hydrogenase and phosphoglycerate kinase. Estimation of en- Sephadex G-200 column, is consistent with the previous zyme activities and absorbance measurements were effected finding that these enzymes may occur under a state of high with a six-sample Pye Unicam 8610 spectrophotometer con- molecular mass [ll, 18 -231. The fractions of the void volume nected to an Apple I1 microcomputer. A unit of enzyme ac- that contain the five enzyme activities generate, upon tivity is defined as the amount of enzyme which transforms hydroxyapatite chromatography, two peaks, A and B, each 1 Fmol /min under the conditions of the assay. of which contains the same five enzyme activities (Fig. 1 B). The ratios of the various enzyme activities remain approx- Determination of molecular mass and homogeneity imately constant across each of the two activity peaks. Two of the complex of these activity ratios are given as an inset in Fig. 1B and some other values are given in the legend of the figure. The Molecular mass estimations were effected by molecular phosphoglycerate kinase activity, however, is too low to be sieve chromatography through Sephacryl S-300, by analytical determined in every fraction across the elution profile. The ultracentrifugation and by slab gel electrophoresis. Sephacryl reason for this low activity is unknown at the moment but 5-300 columns (2.6 x 90 cm) were calibrated with proteins might be due to a poor accessibility of the enzyme's active site of known relative molecular mass (thyroglobulin 669 kDa, within the complex to the bulk substrate. ferritin 449 kDa, catalase 240 kDa, aldolase 147 kDa, bovine The results of the hydroxyapatite chromatography are serum albumin 68 kDa, peroxidase 50 kDa, cytochrome c reminiscent of the findings of Nicholson et al. [24], which 12.5 kDa, aprotinin 6.5 kDa) using buffer B [14]. Analytical showed the existence of two fractions of phosphoribulokinase ultracentrifugation experiments were performed with a activity extracted from the green alga Scenedesmus obliquus. Beckman ultracentrifuge model E equipped with an in- Table 1 shows the respective amounts of the various enzyme terferometric system for molecular mass determinations ac- activities present in the fractions of the two peaks A and B. cording to the method of Yphantis [15] and ultraviolet In order to test the purity of these multienzyme complexes, or schlieren optics for sedimentation patterns. Slab gel electrophoresis was performed in polyacrylamide gradient electrophoresis under denaturing conditions was conducted gels (4-30%) or under native conditions. As shown in Fig. 2, as in [16]. Under non-denaturing conditions the experimental peak A yields a single band with a molecular mass of 449 kDa, protocol proposed by Pharmacia was followed. Protein con- but fractions of peak B contain two molecular entities with centrations were determined by the dye-binding assay of molecular mass of 449 kDa and 669 kDa respectively. These Bradford [17]. results strongly suggest that peak B contains the same multienzyme complex as peak A plus an additional polypep- tide. The homogeneity of the multienzyme complex A may be RESULTS verified by analytical centrifugation. Results of Fig. 3A show In the course of elution of DEAE-Trisacryl columns, the that this complex is indeed homogeneous. A schlieren profile fractions that contain phosphoribulokinase also contain of this fraction is also symmetrical thus giving additional ribose-phosphate isomerase, ribulose-bisphosphate carbox- support to this conclusion (Fig. 3 B). ylase/oxygenase, glyceraldehyde-phosphate dehydrogenase In order to define their composition, electrophoresis of and phosphoglycerate kinase. Despite the large differences of these protein complexes was performed in 9% SDS/poly- their molecular mass (spanning from 50 kDa to 550 kDa), acrylamide gels (denaturing conditions). The results obtained enzyme activities coelute in the void volume of the Sephadex are shown in Fig. 4. G-200 column (Fig. 1 A). A second peak of phosphoribulo- Fractions of peak A show the two subunits of ribulose- kinase activity is obtained later on, during the process of bisphosphate carboxylase/oxygenase, the subunits of phos- elution, and does not contain the other enzyme activities. The phoribulokinase and of glyceraldehyde-phosphate dehydro- presence of glyceraldehyde-phosphate dehydrogenase and genase. The amount of ribose phosphate isomerase and 440

I 50 50. 5 51 51. 5 B r2(crnI2

Fig. 3. (A) Equilibrium sedimentation analysis of complex A at pH 7.5. (B) Sedimentation patterns (schlieren optics) of the jive-enzyme complex. (A) The protein concentration was 1.2 mg/ml. The rotation speed was 9700 rev./min. Column lengths were 3 mm. Temperature 20°C and experiments were run for 48 h. J, the fringe displacement. Buffer used was 100 mM phosphate, 2 mM EDTA, 0.15 M NaCI, 10 mM cysteine, 10% pH 7.5. The molecular mass obtained was 491.3 + 5.7 kDa. (B) Migration is from right to left. Bar angle is 60°C. Photographs were taken every 4 min. Protein concentration was 5.8 mg/ml in buffer B containing glycerol 5%. Photographs show a single symmetrical peak phosphoglycerate kinase is such that no bands pertaining to The multienzyme complex of 536 kDa appears homo- those enzymes are detected. For ribose-phosphate isomerase geneous on analytical centrifugation. By equilibrium sedimen- this is not surprising, since we have found that on tation one finds a molecular mass of 520 kDa, which is consis- polyacrylamide gel, very large amounts (above 30 pg) of this tent with the value of 536 kDa given above. In sedimentation enzyme are needed in order to detect a band by Coomassie velocity studies the pattern appears symmetrical (Fig. 3 B). blue. Fractions of peak B display basically the same pattern Analysis of subunit composition of this complex allows one as those of peak A, except that a band of 65 kDa molecular to detect the subunits of ribulose-bisphosphate carboxylase/ mass is present in the fractions of peak B, but not of the oxygenase, glyceraldehyde-phosphate dehydrogenase, phos- peak A. This 65-kDa protein presents the characteristic of not phoribulokinase and the polypeptide of 65 kDa. being stained by silver dyes (data not shown). Although we Dithiothreitol has been reported to promote the depoly- still do not know the function of this protein, analysis of the merization of a ‘high-molecular-mass form of phosphoribulo- literature suggests that it may correspond to a manganese- kinase [21, 221. This reductant has, therefore, been used to protein possibly involved in oxygen evolution [25]. It thus promote dissociation of ribulose-bisphosphate carboxylase/ appears quite likely that the complex present in the fractions oxygenase from the rest of the complex and to show whether of peak B is also present in the fractions of peak A, but the enzyme obtained in that way may have a molecular mass associated with the 65-kDa polypeptide. different from that of the so-called ‘native’ enzyme, namely If fractions contained in the void volume of Sephadex 550 kDa. Upon treatment of the complex with dithiothreitol G-200 filtration are submitted to Sephacryl S-300 chro- and molecular sieving through Sephacryl, several fractions matography, two peaks are obtained upon elution (Fig. 5). In are obtained (Fig. 6). The first contains a complex of lo3 kDa peak 1 the five enzyme activities are detected as a molecular totally devoid of activity. The second protein peak has entity of 536 kDa. Moreover, under these conditions the ac- ribulose-bisphosphate carboxylase activity, but the corre- tivity of phosphoribose isomerase, phosphoribulokinase and sponding molecular mass is 420 kDa instead of 550 kDa. glyceraldehyde-phosphate dehydrogenase are greatly en- The 65-kDa polypeptide is also detected in the same hanced by preincubation with dithiothreitol. A second protein fraction. peak is also detected on the elution profile that pertains to a The rather low molecular mass of this form of ribulose- molecular mass of 190 kDa and contains ribose-phosphate bisphosphate carboxylase/oxygenase strongly suggests that isomerase and phosphoribulokinase but lacks ribulose-bis- this enzyme may exist with a structure different from the phosphate carboxylase/oxygenase, phosphoglycerate kinase classical ABBBquaternary structure. This 420-kDa form con- and glyceraldehyde-phosphate dehydrogenase activities. taining the 65-kDa polypeptide resembles, however, another 44 1

0 0.5 1 Rf

Fig. 4. (A) 9% SDS/poIyacrylarnide gel; (B) gelcalibration curve. (A) From left to right: track 1, 10 pg 6;track 2, 10 pg rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; track 3, 30 pg spinach phosphoriboisomerase; track 4, 7 pg lactate dehydrogenase; track 5, 10 pg pyruvate kinase; track 6, 15 pg pure spinach phosphoribulokinase; track 7, 50 pg complex B; track 8, mixture of 20 pg phosphoriboisomerase, 10 pg glyceraldehyde-3-phosphatedehydrogenase, 10 pg phosphoribulokinase; track 9, 45 pg complex A. (B) SDS/ polyacrylamide gel calibration curve: phosphorylase b (I), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (4), lactate dehydrogenase (5), pyruvate kinase (2), spinach ribose-phosphate isomerase (6), spinach phosphoribulokinase (3) were used as markers. Arrows indicate the position of polypeptides found in complex A (b, c, d, e) and complex B (a, b, c, d, e). We have found for a, b, c, d and e molecular masses of 65,55,40,38 and 36 kDa. In the bottom of the gel for complexes A and B, a small polypeptide of 14 kDa, corresponding to the small ribulose- bisphosphate carboxylase/oxygenase subunit, was also found. This polypeptide is not represented in the calibration curve because of the poor resolution of small-molecular-mass polypeptides in 9% SDS gel

form of this enzyme isolated from blue-green algae, which techniques, is smaller than the value (904 kDa) one would shows a molecular mass of 450 kDa [26], and another form expect from the sum of the molecular masses of the individual from Rhodopseudomonas palustris and sphaeroides that shows enzymes. A likely explanation of this apparent discrepancy is a molecular mass of 360 kDa [27, 281. The other fractions that the quaternary structures of these enzymes within the contain a complex of 130 kDa molecular mass which has complex are different from the ones they have when isolated phosphoribulokinase activity but has no ribulose-bis- in free solution. A number of arguments seem to support this phosphate carboxylase/oxygenase. It thus appears probable contention. It is well known [29, 301 that the large subunit of that the complex present in the first peak is an artefactual ribulose-bisphosphate carboxylase/oxygenase is highly hy- molecular aggregate resulting from a - inter- drophobic and insoluble in the absence of the small subunit. It change. The interesting result that emerges from these data is is probable that, depending on its microenvironment, different that, after a dithiothreitol treatment, ribulose-bisphosphate interactions with different enzyme may stabilize this carboxylase may occur with a molecular mass and an hydrophobic subunit. The result that dithiothreitol treatment aggregation state different from that of the so-called native promotes a partial depolymerization of the complex, which enzyme. releases an enzyme form that retains its activity, is consistent To show that the complex of these five enzymes of the with this view. A complex made up of four pairs of heavy and Calvin cycle represents a functional entity, ribose 5-phos- light subunits of ribulose-bisphosphate carboxylase/oxyge- phate, the substrate of the first reaction, was mixed with the nase (54 kDa+ 14 kDa) instead of eight, one subunit of complex in the presence of ATP, bicarbonate and NADH, phosphoribulokinase (42 kDa) instead of two, one pair of substrates required for later steps, and the progress of the subunits (39 kDa + 36 kDa) of glyceraldehyde-phosphate de- final reaction was monitored by measuring the production of hydrogenase instead of two, one of phospho- NAD+. The results (Fig. 7) show that NADH is continuously (50 kDa) and one subunit of ribose-phos- oxidized by the complex, thus implying that the sequence of phate isomerase (30 kDa) instead of two, would nicely fit the the five enzyme reactions was indeed taking place. observed molecular mass if the complex is associated with the Moreover, the steady-state velocity of appearance 65-kDa polypeptide. Then the overall molecular mass would at saturating concentrations of ribose phosphate, ATP and be 542 kDa, which is quite compatible with the observed NADH was identical, within experimental error, to the rate values of 536 kDa and 520 kDa. The presence of a 65-kDa of oxidation of NADH at saturating glycerate 3-phosphate, polypeptide chain could also be a point of interest as it re- coenzyme and ATP concentrations. This result suggests the sembles a manganese-protein from the thylakoid membrane existence of channelling within this multienzyme complex. [25].Thus it is attractive to speculate that the 65-kDa polypep- tide corresponds to this thylakoid protein, in which case it DISCUSSION could serve as an anchor for the complex we have described. An important question raised by the present results is that If this were the case, the chloroplast multienzyme complex the molecular mass of the complex, determined by different would be attached to the thylakoid, essentially as the Krebs 442

2. 0 A 1 0.6 0.25 I 1. 5 1

A Ti.0 3

0. 5

0 150 200 250 300 350 Elution volume (mll 150 200 250 300 3 50

2. 5 B Elution volume (ml) Fig. 6. Dissociating effect of dithiothreitol. Gel filtration of Sephacryl S-300 peak I on Sephacryl S-300 after dithiothreitol treatment. A 2. 0 K fraction of concentrated S-300 peak I was reduced with 50mM dithiothreitol for 1 h at 30°C and then submitted to a second gel filtration using buffer B containing 5 mM dithiothreitol instead of 10 mM cysteine. Ribulose-bisphosphate carboxylase/oxygenase ac- tivity (+-+) and phosphoribulokinase activity (0-0) were monitored as described in Materials and Methods. The arrows indi- cate the predicted positions as indicated in Fig. 1 A

3. 5 4 4. 5 5 5. 5 6 Log (Mr)

Fig. 5. (A) Molecular sieving of the ‘void-volume Sephadex G-200 ,fraction’on Sephacryl S-300. (B) Estimation uf the molecular mass of the complex. (A) The different enzyme activities were monitored as described in Materials and Methods. (0-0) Activated phos- phoribulokinase, (0-0) ribose-phosphate isomerase, (A-A) glyceraldehyde-3-phosphate dehydrogenase, (0-0) ribulose-bis- phosphate carboxylasc/oxygenase. The arrows indicate the predicted positions for elution of the isolated enzymes as follows: (1) ribulose- bisphosphate carboxylase/oxygenase, (2) glyceraldehyde-3-phosphate dehydrogenase, (3) phosphoribulokinase, (4) ribose-phosphate iso- merase, (5) phosphoglycerate kinase. Column flow rate was 15 ml/h. The activity ratios, calculated in relation to activated phospho- 1.3 ‘ I ribulokinase, can be considered constant within the expcrimental 0 5 10 15 20 25 error value across peak I. Thus, the activity ratios for ribose-phos- Time(rnin) phate isomerase at 224 ml, 234 ml and 244 ml are respectively 0.16, 0.16,0.33; for glyceraldehyde-3-phosphate dehydrogenase at 214 ml, Fig. I. Functionality of the 520-kDa complex. The multienzyme 234 ml and 244 ml thcy are respectively, 0.2, 0.29, 0.21; for ribulose- complex was preincubated for 15min in the presence of 50mM bisphosphate carboxylase/oxygenasc at 224 ml, 234 ml and 244 ml glycylglycine, 0.5 mM EDTA, 10 mM MgZt, 5 mM dithiothreitol they are, respectively, 0.16,0.31,0.36. (A) Estimation of the molecular and 50 mM sodium bicarbonate. After this prcincubation, the activity mass was performed by molecular sieving through a calibrated of thc complex was measured at pH 7.7 by adding the following (final Sephacryl S-300 column by following the technique of Whitaker [14]. concentrations): 1 mM ribose Sphosphate, 1 mM ATP, 0.25 mM Aprotinin (l), cytochrome c (2), peroxidase (3), bovine serum albumin NADH, 5 units triose-phosphate isomerase and 11 units gly- (4), aldolase (9,catalase (6), ferritin (7), thyroglobulin (8) were used cerophosphate dehydrogenase in a final volume of 1 ml. The as markers. The arrow indicates the position of Sephacryl S-300 peak multienzyme complex concentration used was 0.3 pM. A control, in I, which corresponds to a molecular mass of 536 kDa which ribose 5-phosphate was omitted, gave no decrease of absorbance indicating no interfering NADH oxidase activity (other details are as described in Materials and Methods) cycle multienzyme complex is attached to the inner mitochondria1 membrane [4]. The main point of this study is related to the functionality Some results presented above are quite consistent with of the multienzyme complex. The existence of a complex of previous proposals. For instance, ribulose-bisphosphate car- this sort in chloroplasts may represent a functional advantage boxylase of pea is associated with ribose-phosphate isomerase as it allows chanelling of reaction intermediates of the Calvin and phosphoribulokinase activities [7]. Moreover the forms cycle from one site to another, thus preventing diffusion of of phosphoribulokinase and glyceraldehyde-phosphate de- these intermediates in the bulk phase. Results presented in hydrogenase of high molecular mass (550 kDa [21, 221 and this report suggest that this release of intermediates does not 600 kDa [18,20] respectively) that have been called ‘regulatory occur to a significant extent, as the velocity of the last step of forms’ may either represent an aggregated state of the same the reaction sequence at saturating substrate concentration is enzyme molecules or a multienzyme complex similar to the identical to that of the overall process at saturating ribose- one studied in this report. phosphate concentration. Although the existence of chan- 443 nelling cannot yet be taken as proved, it is suggested by these 14. Whitaker, J. R. (1963) Anal. Chem. 35, 1950-1953. results. Further studies are now under way to investigate it 15. Yphantis, D. A. (1964) 3, 297-317. further and to study its dynamics. 16. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 17. Bradford, M. M. (1976) Anal. Biochem. 72,248-252. The expert technical assistance of Mrs Mireille Rivikre and Mr 18. Yonuschol, G. R., Ortwerth, B. J. & Koeppe, 0. J. (1970) J. Bid. Paul Sauve is deeply appreciated. Thanks are due to Mr A. Ribas for Chem. 245,4193-4198. having grown the spinach. This work was supported in part by 19. Pupillo, P. & Piccari, G. G. (1973) Arch. Biochem. Biophys. 154, Biologic. Muleculuire Egbtale du Centre National de la Recherche 324 - 33 1. Scientijique, Action Thematique Programmbe. 20. Pupillo, P. & Piccari, G. G. (1975) Eur. J. Biochem. 51,475-482. 21. Ward-Aswapati, O., Kemble, R. J. & Bradbeer, J. W. (1980) Plant Physiol. 66, 34 - 39. REFERENCES 22. Bradbeer, J. W., Riiffer-Turner, M. E., Deferre, E. J., Wara 1. Fulton, A. B. (1982) Cell 30, 345-347. Aswapati, 0. & Kemble, R. J. (1981) IV, regu- 2. Clegg, J. S. (1984) Am. J. Physiol. 246, R133-151. lation of carbon (Akoyunoglou Balaban, G., ed.) 3. Tompa, P., Bar, J. & Batke, J. (1986) Eur. J. Biochem. 159, 117- pp. 389 - 394, International Science Services, Philadelphia. 124. 23. Wolosiuk, R. A. & Buchanan, B. B. (1978) Arch. Biochem. Bio- 4. Robinson, J. B. & Srere, P. A. (1985) J. Biol. Chem. 260,10800- phys. l#Y, 97-101. 10805. 24. Nicholson, S., Easterby, J. S. & Powls, R. (1987) Eur. J. Biochem. 5. Welch, G. R. & Clegg, J. S. (eds) (1987) The organization of cell 162,423-431. metabolism, Plenum Press, New York. 25. Spector, M. & Winget, G. D. (1980) Proc. Nut1 Acad. Sci. USA 6. Keleli, T. & Ovadi, J. (1987) Curr. Top. Cell. Regul., in the press. 77,957 - 959. 7. Sainis, J. K. & Harris, G C. (1986) Biochem. Biophys. Res. 26. Tabita, F. R., Stevens, S. E. Jr & Gibson, J. (1974) J. Bacteriol. Commun. 139,947-954. 125, 531 - 539. 8. Anderson, L. E. (1987) FEBS Lett. 212,45-48. 27. Anderson, L. E., Price, G. B. & Fuller, R. C. (1968) Science 9. Jensen, R. G. & Bassham, J. A. (1966) Proc. Natl Acad. Sci. USA (Wash. DC) 161,482-484. 56,1095-1101. 28. Gibson, J. L. & Tabita, F. R. (1977) J. Biol. Chem. 252, 943- 10. Porter, M. A,, Milanez, S., Stringer, C. D. & Hartman, F. C. 949. (1986) Arch. Biochem. Biophys. 245,14-23. & 11. Pawlizki, K. & Latzko, E. (1974) FEES Lett. 42,285-288. 29. Jordan, D. B. Chollet, R. (1985) Arch. Biochem. Biupizys. 236, 12. Lavergne, D. & Bismuth, E. (1973) Plant. Sci. Lett., 229-236. 487 - 496. 13. Pierce, J., Lorimer, G. H. & Reddy, G. S. (1986) Biochemistry 25, 30. Andrews, T. J. & Lorimer, G. H. (1985) J. Bid. Chem. 260,4632- 1636- 1644. 4636.