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The physiological significance of aggregation phenomena of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts
186
Biochimica et Btophvst
Elsevier BBA 32362
T h e physiological significance of aggregation p h e n o m e n a of N A D P - d e p e n d e n t glyceraldehyde-3-phosphate d e h y d r o g e n a s e from spinach chloroplasts Lois M. Levy and Graham
F. B e t t s
School of Biological Sciences, Queen Mary College, Mile End Road, London, El 4NS (U. K.)
(Received May 11th, 1985)
Key words: Glyceraldehyde-3-phosphatedehydrogerase kinetics; NADP(H) activation; RNA contamination: (Spinach chloroplast)
it has been shown by deLooze and Wagner (Physiol. Plant. 57 (1983) 243-249) that the association/ dissociation phenomena of NADP-dependent D-glyceraldehyde-3-phosphate oxidoreductase (phosphorylating) (D-glyceraldehyde-3-phosphate:NADP + oxidoreductase (phosphorylating), EC 1.2.1.13) from Chenopodium rubrum is dependent on the presence of an RNA fraction. We have shown the same is true for enzyme from the more popular source material Spinacia oleracea. However, extraction of enzyme from previously isolated chloroplasts show that the responsible RNA fraction is not present with the enzyme in the chloroplasts. It is therefore hard to see the physiological relevance of the RNA effect. Giyceraldehyde-3phosphate dehydrogenase free from the RNA fraction shows none of the NAD/NADP-dependent activity changes and non-Michaelis-Menten kinetic behaviour which have been a feature of several previous reports using enzyme which may not have been RNA-free. Introduction Several papers dealing with NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (Dglyceraldehyde-3-phosphate: N A D P + oxidoreductase (phosphorylating), EC 1.2.1.13) from higher plant chloroplasts have been concerned with reversible protein aggregation phenomena affected by various natural small molecules including the substrates of the reaction [2-7]. Impetus for these studies has been provided by reports that the same molecules that affect enzyme aggregation also affect activity, those molecules promoting enzyme dissociation tending to activate NADP(H)-dependent activity [2,3,8,9]. This clearly raises the possibility that activity (and aggregation state) may not be constant in vivo, but may change as a result of changing concentration of effectors. This would provide a possible control mechanism in the switch of metabolism upon the light/dark transition. DeLooze and Wagner [1] introduced a further
consideration following an earlier clue by Cerff [6]. They showed that association of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Chenopodium rubrum to a molecular weight of at least 600000 was promoted by an RNA-rich fraction which copurified with the enzyme until separated from it by molecular exclusion chromatography in the presence of N A D P +. The aggregation phenomena of the enzyme thereafter was dependent on whether the RNA fraction was added back or not. We have studied three aspects of this problem. (1) Do the phenomena reported by deLooze and Wagner [1] for the enzyme from Chenopodium also occur in glyceraldehyde-3-phosphate dehydrogenase isolated from the more popular source material Spinacia oleracea? (2) Does the effect of the RNA have physiological significance or is it an artifact of the extraction procedure? (3) If the effect of RNA is an artifact, what are
0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
187 the kinetic properties of enzyme from which the R N A fraction has been rigorously removed? Materials and Methods
Enzyme purification (a) Whole leaf preparation. This was by the method of Pupillo and Faggiani [10] except for the following: (i) Material was S. oleracea obtained from market; (ii) the first ammonium sulphate fractionation was between 40-60% saturation; (iii) the DEAE-cellulose chromatography was omitted and the resuspended acetone precipitate was precipitated with ammonium sulphate to 75% saturation, collected by centrifugation and resuspended in 100 mM Tris-HC1/5 mM E D T A / 2 mM dithiothreitol/100 mM KC1, with or without 0.2 mM N A D P + (pH 8.0). (The DEAE-cellulose chromatography was omitted because the preceding acetone fractionation gave a greater and the subsequent DEAE-cellulose a smaller increase than those found by Pupillo and Faggiani [10]). Specific activity of a routine preparation was 11 ~mol N A D P H oxidised per min per mg protein in the standard assay below. Enzyme was stored at - 1 8 ° C until used. (b) From previously isolated chloroplasts. Chloroplasts were isolated from 400 g of leaves by the method of Charles and Halliwell [11] and were 60% intact as judged by the ferricyanide reduction test [12]. Intact washed chloroplasts were burst by suspending in 50 mM H e p e s / 2 mM E D T A / 2 mM dithiothreitol (pH 7.5 with KOH), incubated 15 min at 0°C and centrifuged at 30000 g for 30 rain at 4°C. Supernatant containing enzyme was decanted off and concentrated to 1 ml using Minicon-B concentrating units (Amicon Corp., Danvers, MA, U.S.A.). 60 units of enzyme activity were obtained from the 400 g of leaf.
Chromatography Gel chromatography was carried out at 4°C using a Sephacryl S-300 column (1.3 × 39.5 cm). The column was developed by downward flow adjusted to 12 m l / h by the use of a peristaltic pump. Samples were applied to the column in a volume of 1 ml. The column was equilibrated with 50 mM T r i s - H C l / 5 mM E D T A / 2 mM dithioth-
reitol (pH 7.5) plus 0.2 mM N A D + or N A D P +. The molecular weight markers catalase (232000), and lactate dehydrogenase (145 000) were used to calibrate the column. The void volume was determined using Blue Dextran 2000.
Enzyme assay N A D P H oxidation was followed at 340 nm using a Varian DMS100 spectrophotometer. The reaction mixture contained, in 1 ml total volume, 100 mM Tris-HCl (pH 8.0), 4.5 mM 3-phosphoglycerate, 10 mM 2-mercaptoethanol, 8 mM MgSO 4, 2 mM ATP, 1 mM EDTA, 1.6 units phosphoglycerate kinase, 0.1 mM N A D P H , and glyceraldehyde-3-phosphate dehydrogenase (up to 0.015 units). For routine assays the reaction was started by the addition of 3-phosphoglycerate, after 2 min equilibration of all other components at 25°C. In specified cases, the reaction was started with enzyme. For K,,, determinations the N A D P H concentration is varied between 0.008 and 0.3 mM. Rapid mixing and stopped flow experiments were conducted using a Durrum 13000 Spectrophotometer (Durrum Instruments, Palo Alto, CA, U.S.A.). Enzyme was diluted into 100 mM TrisHC1, 1 mM EDTA, 10 mM mercaptoethanol (pH 8.0) and placed in one syringe. This was mixed with the contents of the second syringe to give conditions identical to those in the standard assay, except that enzyme concentration was usually 5fold higher and phosphoglycerate kinase was 10fold higher.
Preincubation experiments For preincubation experiments, enzyme (either from chloroplast or whole leaf preparations) was diluted into 50 mM Tris-HC1/5 mM E D T A / 2 mM dithiothreitol (pH 7.5) containing either 0.2 mM N A D + or 0.2 mM NADP(H). The enzyme solution was incubated at 25°C and aliquots taken for assay immediately and every 10 min thereafter for a period of 1 h. RNAase for hydrolysis of void volume fraction from Sephacryl S-300 columns was heat treated to remove DNAase activity. E D T A concentration in the void volume fractions was lowered to less than 0.05 mM by successive concentrations on Minicon-B concentrators and redilution into 0.015 M sodium citrate/0.15 M NaCI (pH 7.0). The
188 R N A a s e d i g e s t i o n was c o m p l e t e d in this b u f f e r by the a d d i t i o n o f 10 /~g R N A a s e a n d i n c u b a t e d at r o o m t e m p e r a t u r e for 30 min.
Reagents All r e a g e n t s w e r e of a n a l y t i c a l grade. E n z y m e substrates, yeast p h o s p h o g l y c e r a t e kinase, m o l e c u lar w e i g h t m a r k e r s a n d R i b o n u c l e a s e A t y p e X I I - A are o b t a i n e d f r o m S i g m a C h e m i c a l C o m p a n y . S e p h a c r y l S-300 a n d Blue D e x t r a n w e r e o b t a i n e d from Pharmacia.
Results Fig. 1 d e m o n s t r a t e s that N A D P - d e p e n d e n t glyceraldehyde-3-phosphate d e h y d r o g e n a s e isol a t e d f r o m the e n t i r e l e a f of s p i n a c h s h o w s the NAD+-promoted aggregation and NADP+-pro m o t e d d i s a g g r e g a t i o n p r e v i o u s l y r e p o r t e d [1-6]. Fig. 2 s h o w s t h a t w h e n the d i s s o c i a t e d e n z y m e f r o m Fig. 1 is r e c h r o m a t o g r a p h e d in the p r e s e n c e
o f N A D +, the r e a g g r e g a t i o n d e p e n d s on the prese n c e of m a t e r i a l e l u t e d w i t h the v o i d v o l u m e f r o m the N A D P + - r u n c o l u m n . T h e v o i d v o l u m e f r a c t i o n of S e p h a c r y l S-300 c o l u m n s r u n in the p r e s e n c e of N A D P + has a r a t i o o f a b s o r b a n c e 260 n m / 2 8 0 n m of 2.3, c h a r a c t e r i s tic o f n u c l e i c acids r a t h e r t h a n p r o t e i n s . T h e ability of this v o i d v o l u m e m a t e r i a l to p r o m o t e the a g g r e g a t i o n of g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e is d e s t r o y e d on t r e a t m e n t w i t h R N A a s e . All the a b o v e results w i t h s p i n a c h are essentially the s a m e as t h o s e of d e L o o z e a n d W a g n e r [1] w o r k i n g with Chenopodium. W e h a v e a p p r o a c h e d the q u e s t i o n of the physiological s i g n i f i c a n c e of these effects b y s u p p o s i n g that the R N A r e s p o n s i b l e m a y n o t e v e n be in the c h l o r o p l a s t s w i t h the g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e b u t o n l y a s s o c i a t e w i t h it u p o n cell a n d c h l o r o p l a s t b r e a k a g e . If this is the case, enz y m e s e x t r a c t e d f r o m c h l o r o p l a s t s p r e v i o u s l y isol a t e d i n t a c t f r o m the cell w o u l d n o t h a v e the R N A
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Fig. 1. Effect of N A D / N A D P on the elution profile of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Sephacryl S-300 columns. 1 ml of partially purified enzyme stored at - 18°C was thawed and applied to a Sephacryl S-300 column (1.3 × 39.5 cm) previously equilibrated with 50 mM Tris-HC1/5 mM EDTA/2 mM dithiothreitol/0.2 mM NAD ÷ (pH 7.5). Flow rate was 12 ml/h. 12 ml of eluate was rejected, then fractions of 1.1 ml were collected. Fractions 7-14 were collected, concentrated to 1 ml and rechromatographed on the same column but previously equilibrated with the above buffer containing 0.2 mM NADP +. • •, NAD+; or o - - -- - - o , NADP + in the chromatography buffer.
Fig. 2. Effect of high-molecular-weight fraction on the elution profile of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase. Enzyme was stored and chromatographed in an identical manner to Fig. 1, first in buffer containing 0.2 mM NADP + in place of NAD+. Fractions 8-12 were collected (4 ml) and called void-volume material. Enzyme activity eluted between fractions 15 and 23 and was collected as 8 ml. 4 ml of this was concentrated to 1 ml and rechromatographed on the same column equilibrated with buffer containing 0.2 ml NAD ® in place of NADP ÷. This elution profile is labelled o - - - - --o. 4 ml of enzyme remaining from the NADP ÷ chromatography was added back to 2 ml of the void volume material. This was then concentrated to 2 ml and 1 ml of this was rechromatographed on a column equilibrated with buffer containing 0.2 mM NAD ÷ in place of NADP ÷. This is the elution profile •
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189
present and would therefore exhibit altered aggregation phenomena. Fig. 3 shows an identical experiment to Fig. 1 except that enzyme has been extracted from previously isolated chloroplasts. It is clearly seen that even in the presence of N A D ÷ the bulk of the glyceraldehyde-3-phosphate dehydrogenase exists as a unit having a molecular weight of approx. 300000 and only a small part aggregates to the species of 600 000 or more. If, however, enzyme from chloroplasts is first mixed with the void volume fraction from a whole leaf preparation chromatographed on Sephacryl S-300 in the presence of N A D P +, then all activity aggregates in the presence of N A D ÷ to a species of molecular weight above 600000. The void volume fraction from the column of Fig. 3 run in the presence of N A D P ÷ has a ratio of absorbance 260 n m / 2 8 0 nm of 1.0, indicating a low concentration of RNA relative to protein. Fig. 4 shows that this void volume material fails to induce the NAD-dependent reassociation of enzyme prepared from whole leaves and previously disaggregated on a Sephacryl column run in the presence of N A D P ÷, whereas in an otherwise identical experiment void volume material from
whole leaf preparations induces NAD-dependent reassociation. The experiment entirely analogous to that of Fig. 4, but using dissociated glyceraldehyde-3phosphate dehydrogenase from previously isolated chloroplasts, gives identical elution profiles. Several groups have reported that NADP(H) which promotes the disaggregation of glyceraldeh y d e - 3 - p h o s p b a t e d e h y d r o g e n a s e activates NADP(H)-linked activity [2,3,9] by drastically reducing the K m for N A D P ( H ) [2]. The N A D ( H ) which promotes enzyme aggregation reverses this effect. Moreover non-Michaelis-Menten kinetic behaviour has been sometimes [2,5,9,13] but not always [14] reported. We have surmised that if aggregation phenomena are no longer seen in the absence of endogenous RNA, the associated changes in kinetic constants may also be absent. We have therefore begun the kinetic investigation of NADP-dependent glyceraldehyde-3-phosphate
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Fig. 3. Effect of N A D / N A D P on the elution profile of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase obtained from previously isolated chloroplasts. 1 ml of enzyme prepared from chloroplasts (see Materials and Methods) was chromatographed in an identical manner to Fig. 1. Enzyme chromatographed in buffer containing ([3 [3) 0.2 mM N A D +, (0-- ----0) 0.2 mM N A D P +,
was chromatographed on Sephacryl S-300 as in Fig. 2 in buffer containing 0.2 mM N A D P +, Fractions 8 - 1 2 were collected and called void volume material. Enzyme activity eluted between fractions 15 and 23 (8 ml) and was collected. An identical chromatography used 1 ml of enzyme from previously isolated chloroplasts and void volume material only was collected between fractions 8-12. 2 ml of enzyme from the first column were mixed with 1 ml of void volume material from either the first column (whole leaf preparation) or the second column (chloroplast preparation). Both samples were concentrated to 1 ml and chromatographed in buffer containing 0.2 mM N A D ÷. Elution profiles of enzyme with void volume material from (A A) whole leaf (m-- - - --m) isolated chloroplasts.
190
dehydrogenase from which has been removed the high-molecular-weight fraction containing RNA. We have used enzyme extracted from previously isolated chloroplasts or enzyme from whole leaf preparations which have been chromatographed on Sephacryl S-300 in the presence of N A D P + to remove the R N A fraction eluting with the void volume. Preincubation of enzyme at 25°C in the chro-
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matography buffer containing either 0.2 mM N A D + or 0.2 mM N A D P H leads to enzyme having identical activity in the normal reaction mixture when glyceraldehyde-3-phosphate dehydrogenase is added last to initiate the reaction. We have tested to see whether the failure to observe a difference in the activity between enzyme which has been presoaked in N A D * or N A D P H is due to activation by the N A D P H or A T P [3,9] of the assay mixture within the mixing time of a normal spectrophotometric experiment (approx. 10 s). Mixing time was reduced by using a rapid mixing and stopped flow spectrophotometer. Enzyme incubated in 0.2 mM N A D + and rapidly mixed to give standard assay conditions show no evidence of rate acceleration over the period 5 ms to 10 s. A previous study [2] has reported that the main activating effect of preincubation with N A D P ( H ) is to lower the K m for NADP(H). There is the possibility that our failure to observe any activating effects of N A D P ( H ) is due to the use during assay of near-saturating concentrations of N A D P H . Fig. 5 shows that with enzyme devoid of the high-molecular-weight RNA fraction, the K,1 for N A D P H is no different whether the enzyme is preincubated in N A D + or N A D P +. Typical Michaelis-Menten kinetics are observed in both cases.
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Fig. 5. Substrate saturation kinetics of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase previously equilibrated in (A) 0.2 m M N A D P +, (B) 0.2 m M N A D +. Glyceraldehyde-3-phosphate dehydrogenase from a whole leaf preparation was chromatographed on Sephacryl S-300 in the presnce of 0.2 mM N A D P + to remove void-volume material containing R N A and equlibrated against the same buffer as in Fig. 1 containing either 0.2 m M N A D + or 0,2 m M N A D P +. Samples were assayed in 100 m M Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol, 8 m M MgSO 4, 2 mM ATP, 4.5 mM 3-phosphoglycerate, 1.6 units phosphoglycerate kinase, 0.015 units of glyceraldehyde-3-phosphate dehydrogenase and N A D P H as shown in a total volume of l ml. Line fitted by the method of Johansen and Lumry [15]. K,,, for N A D P H 25 # M (A) and 28 ,ttM (B).
The implication of several studies on N A D P dependent chloroplast glyceraldehyde-3-phosphate dehydrogenase has been that, in the presence of N A D +, a monomer of molecular weight 150000 aggregates to a tetramer of 600000 [2,7,16,17]. However, if an RNA-rich fraction which itself has a molecular weight of over 600000 induces glyceraldehyde-3-phosphate dehydrogenase in the presence of N A D + to elute from molecular exclusion chromatograms at a molecular weight of at least 600000, there seems little need to hypothesise a polymeric structure to account for the apparent increase in molecular weight. We are uncertain of the significance of the intermediate molecular weight profile of glyceraldehyde-3-phosphate dehydrogenase from isolated chloroplasts when first chromatographed in the
191
presence of NAD + (Fig. 3). It may be thought that an intermediate level of association is due to a low level of contamination of chloroplasts with extrachloroplastic RNA. This idea is supported by the fact that enzyme from previously isolated chloroplasts and chromatographed in the presence of N A D P + (which would free it from RNA eluting in the void volume) shows only the low molecular weight profile when rechromatographed in the presence of NAD +. Moreover, void volume material from chloroplasts preparations run on Sephacryl S-300 in the presence of NADP + has a slight tendency to promote NAD+-dependent reassociation of enzyme which has been freed from RNA (Fig. 4). In studies on enzyme activity we have not used enzyme concentrations as high as that used in chromatography experiments. There is still the possibility that at higher concentrations, enzyme free from nonchloroplast RNA does alter activity as the N A D / N A D P ratio changes. What is true is that these activity changes will be unrelated to association/dissociation phenomena, as Figs. 2 and 4 show none to exist in RNA-free enzyme preparations. The most extensive steady-state kinetic analysis of NADP-dependent chloroplast glyceraldehyde3-phosphate dehydrogenase published to date [14] shows no evidence of the non-Michaelis-Menten behaviour which would be revealed if NADP(H) activation was occurring. It is worth noting that this study [14] was accomplished with an enzyme which experienced, as an integral part of its purification, a molecular exclusion chromatography in the presence of N A D P +. This would have removed the crucial RNA fraction. Moreover, classical Michaelis-Menten behaviour was seen in another study which extracted enzyme from previously isolated chloroplasts [18]. Both of these studies are therefore consistent with the conclusion reached here that this enzyme behaves in a classical Michaelis-Menten fashion with no preactivation by NADP(H) required. In our hands NADP-dependent chloroplast glyceraldehyde-3-phosphate dehydrogenase has never shown non-Michaelis-Menten behaviour with
respect to NADPH, nor does N A D P H activate enzyme previously incubated in NAD +. In view of this study, however, and those of Cerff [14] and Speranza et al. [18], it may be useful to those who obtain such effects to investigate the role of contaminant RNA.
Acknowledgement We wish to thank for their assistance Dr. K. Brocklehurst and Dr. Francis Willenbrock of the Department of Biochemistry, St. Bartholomew's Hospital Medical School, London, in whose laboratory the stopped-flow experiments were carried out.
References 1 deLooze, S. and Wagner, E. (1983) Physiol. Plant. 57, 243-249 2 Pupillo, P. and Piccari, G.G. (1973) Arch. Biochem. Biophys. 154, 324-331 3 Pupillo, P. and Piccari, G.G. (1975) Eur. J. Biochem. 51, 475-482 4 Schwarz, Z., Maretzki, D. and Schonherr, J. (1976) Biochem. Physiol. Pflanzen 170, 37-50 5 Cerff, R. (1978) Eur. J. Biochem. 82, 45-53 6 Cerff, R. (1978) Plant Physiol. 61,369-372 7 Wara-Aswapati,O., Kemble, R.J. and Bradbeer, J.W. (1980) Plant Physiol. 66, 34-39 8 Pawlizki, K.H. and Latzko, E. (1974) FEBS Len. 42, 285-288 9 Wolosiuk, R.A. and Buchanan, B.B. (1976) J. Biol. Chem. 251, 6456 6461 10 Pupillo, P. and Faggiani, R. (1979) Arch. Biochem. Biophys. 194, 581592 11 Charles, S.A. and Halliwell, B. (1981) Cell Calcium 2, 211-224 12 Lilley, R.M.C., Fitzgerald, M.P., Rienits, K.G. and Walker, D.A. (1975) New Phytol. 75, 1-10 13 Ziegler, I., Marewa, A. and Schoepe, E. (1976) Phytochemistry 15, 1627-1632 14 Cerff, R. (1978) Phytochemistry 17, 2061-2067 15 Johansen, G, and Lumry, R. (1961) C.R. Trav. Lab. Carlsberg 32, 185-214 16 Ferri, G., Comerio, G., Iadarola, P., Zapponi, M.C. and Speranza, M.L. (1978) Biochim. Biophys. Acta 522, 19-31 17 Zapponi, M.C., Berni, R., ladarola, P. and Ferri, G. (1983) Biochim. Biophys. Acta 744, 260 264 18 Speranza, M.L., Zapponi, M.C. and Iadorola, P. (1982) ltal. J. Biochem. 31, 22-27
Biochimica et Btophvst
Elsevier BBA 32362
T h e physiological significance of aggregation p h e n o m e n a of N A D P - d e p e n d e n t glyceraldehyde-3-phosphate d e h y d r o g e n a s e from spinach chloroplasts Lois M. Levy and Graham
F. B e t t s
School of Biological Sciences, Queen Mary College, Mile End Road, London, El 4NS (U. K.)
(Received May 11th, 1985)
Key words: Glyceraldehyde-3-phosphatedehydrogerase kinetics; NADP(H) activation; RNA contamination: (Spinach chloroplast)
it has been shown by deLooze and Wagner (Physiol. Plant. 57 (1983) 243-249) that the association/ dissociation phenomena of NADP-dependent D-glyceraldehyde-3-phosphate oxidoreductase (phosphorylating) (D-glyceraldehyde-3-phosphate:NADP + oxidoreductase (phosphorylating), EC 1.2.1.13) from Chenopodium rubrum is dependent on the presence of an RNA fraction. We have shown the same is true for enzyme from the more popular source material Spinacia oleracea. However, extraction of enzyme from previously isolated chloroplasts show that the responsible RNA fraction is not present with the enzyme in the chloroplasts. It is therefore hard to see the physiological relevance of the RNA effect. Giyceraldehyde-3phosphate dehydrogenase free from the RNA fraction shows none of the NAD/NADP-dependent activity changes and non-Michaelis-Menten kinetic behaviour which have been a feature of several previous reports using enzyme which may not have been RNA-free. Introduction Several papers dealing with NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (Dglyceraldehyde-3-phosphate: N A D P + oxidoreductase (phosphorylating), EC 1.2.1.13) from higher plant chloroplasts have been concerned with reversible protein aggregation phenomena affected by various natural small molecules including the substrates of the reaction [2-7]. Impetus for these studies has been provided by reports that the same molecules that affect enzyme aggregation also affect activity, those molecules promoting enzyme dissociation tending to activate NADP(H)-dependent activity [2,3,8,9]. This clearly raises the possibility that activity (and aggregation state) may not be constant in vivo, but may change as a result of changing concentration of effectors. This would provide a possible control mechanism in the switch of metabolism upon the light/dark transition. DeLooze and Wagner [1] introduced a further
consideration following an earlier clue by Cerff [6]. They showed that association of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Chenopodium rubrum to a molecular weight of at least 600000 was promoted by an RNA-rich fraction which copurified with the enzyme until separated from it by molecular exclusion chromatography in the presence of N A D P +. The aggregation phenomena of the enzyme thereafter was dependent on whether the RNA fraction was added back or not. We have studied three aspects of this problem. (1) Do the phenomena reported by deLooze and Wagner [1] for the enzyme from Chenopodium also occur in glyceraldehyde-3-phosphate dehydrogenase isolated from the more popular source material Spinacia oleracea? (2) Does the effect of the RNA have physiological significance or is it an artifact of the extraction procedure? (3) If the effect of RNA is an artifact, what are
0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
187 the kinetic properties of enzyme from which the R N A fraction has been rigorously removed? Materials and Methods
Enzyme purification (a) Whole leaf preparation. This was by the method of Pupillo and Faggiani [10] except for the following: (i) Material was S. oleracea obtained from market; (ii) the first ammonium sulphate fractionation was between 40-60% saturation; (iii) the DEAE-cellulose chromatography was omitted and the resuspended acetone precipitate was precipitated with ammonium sulphate to 75% saturation, collected by centrifugation and resuspended in 100 mM Tris-HC1/5 mM E D T A / 2 mM dithiothreitol/100 mM KC1, with or without 0.2 mM N A D P + (pH 8.0). (The DEAE-cellulose chromatography was omitted because the preceding acetone fractionation gave a greater and the subsequent DEAE-cellulose a smaller increase than those found by Pupillo and Faggiani [10]). Specific activity of a routine preparation was 11 ~mol N A D P H oxidised per min per mg protein in the standard assay below. Enzyme was stored at - 1 8 ° C until used. (b) From previously isolated chloroplasts. Chloroplasts were isolated from 400 g of leaves by the method of Charles and Halliwell [11] and were 60% intact as judged by the ferricyanide reduction test [12]. Intact washed chloroplasts were burst by suspending in 50 mM H e p e s / 2 mM E D T A / 2 mM dithiothreitol (pH 7.5 with KOH), incubated 15 min at 0°C and centrifuged at 30000 g for 30 rain at 4°C. Supernatant containing enzyme was decanted off and concentrated to 1 ml using Minicon-B concentrating units (Amicon Corp., Danvers, MA, U.S.A.). 60 units of enzyme activity were obtained from the 400 g of leaf.
Chromatography Gel chromatography was carried out at 4°C using a Sephacryl S-300 column (1.3 × 39.5 cm). The column was developed by downward flow adjusted to 12 m l / h by the use of a peristaltic pump. Samples were applied to the column in a volume of 1 ml. The column was equilibrated with 50 mM T r i s - H C l / 5 mM E D T A / 2 mM dithioth-
reitol (pH 7.5) plus 0.2 mM N A D + or N A D P +. The molecular weight markers catalase (232000), and lactate dehydrogenase (145 000) were used to calibrate the column. The void volume was determined using Blue Dextran 2000.
Enzyme assay N A D P H oxidation was followed at 340 nm using a Varian DMS100 spectrophotometer. The reaction mixture contained, in 1 ml total volume, 100 mM Tris-HCl (pH 8.0), 4.5 mM 3-phosphoglycerate, 10 mM 2-mercaptoethanol, 8 mM MgSO 4, 2 mM ATP, 1 mM EDTA, 1.6 units phosphoglycerate kinase, 0.1 mM N A D P H , and glyceraldehyde-3-phosphate dehydrogenase (up to 0.015 units). For routine assays the reaction was started by the addition of 3-phosphoglycerate, after 2 min equilibration of all other components at 25°C. In specified cases, the reaction was started with enzyme. For K,,, determinations the N A D P H concentration is varied between 0.008 and 0.3 mM. Rapid mixing and stopped flow experiments were conducted using a Durrum 13000 Spectrophotometer (Durrum Instruments, Palo Alto, CA, U.S.A.). Enzyme was diluted into 100 mM TrisHC1, 1 mM EDTA, 10 mM mercaptoethanol (pH 8.0) and placed in one syringe. This was mixed with the contents of the second syringe to give conditions identical to those in the standard assay, except that enzyme concentration was usually 5fold higher and phosphoglycerate kinase was 10fold higher.
Preincubation experiments For preincubation experiments, enzyme (either from chloroplast or whole leaf preparations) was diluted into 50 mM Tris-HC1/5 mM E D T A / 2 mM dithiothreitol (pH 7.5) containing either 0.2 mM N A D + or 0.2 mM NADP(H). The enzyme solution was incubated at 25°C and aliquots taken for assay immediately and every 10 min thereafter for a period of 1 h. RNAase for hydrolysis of void volume fraction from Sephacryl S-300 columns was heat treated to remove DNAase activity. E D T A concentration in the void volume fractions was lowered to less than 0.05 mM by successive concentrations on Minicon-B concentrators and redilution into 0.015 M sodium citrate/0.15 M NaCI (pH 7.0). The
188 R N A a s e d i g e s t i o n was c o m p l e t e d in this b u f f e r by the a d d i t i o n o f 10 /~g R N A a s e a n d i n c u b a t e d at r o o m t e m p e r a t u r e for 30 min.
Reagents All r e a g e n t s w e r e of a n a l y t i c a l grade. E n z y m e substrates, yeast p h o s p h o g l y c e r a t e kinase, m o l e c u lar w e i g h t m a r k e r s a n d R i b o n u c l e a s e A t y p e X I I - A are o b t a i n e d f r o m S i g m a C h e m i c a l C o m p a n y . S e p h a c r y l S-300 a n d Blue D e x t r a n w e r e o b t a i n e d from Pharmacia.
Results Fig. 1 d e m o n s t r a t e s that N A D P - d e p e n d e n t glyceraldehyde-3-phosphate d e h y d r o g e n a s e isol a t e d f r o m the e n t i r e l e a f of s p i n a c h s h o w s the NAD+-promoted aggregation and NADP+-pro m o t e d d i s a g g r e g a t i o n p r e v i o u s l y r e p o r t e d [1-6]. Fig. 2 s h o w s t h a t w h e n the d i s s o c i a t e d e n z y m e f r o m Fig. 1 is r e c h r o m a t o g r a p h e d in the p r e s e n c e
o f N A D +, the r e a g g r e g a t i o n d e p e n d s on the prese n c e of m a t e r i a l e l u t e d w i t h the v o i d v o l u m e f r o m the N A D P + - r u n c o l u m n . T h e v o i d v o l u m e f r a c t i o n of S e p h a c r y l S-300 c o l u m n s r u n in the p r e s e n c e of N A D P + has a r a t i o o f a b s o r b a n c e 260 n m / 2 8 0 n m of 2.3, c h a r a c t e r i s tic o f n u c l e i c acids r a t h e r t h a n p r o t e i n s . T h e ability of this v o i d v o l u m e m a t e r i a l to p r o m o t e the a g g r e g a t i o n of g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e is d e s t r o y e d on t r e a t m e n t w i t h R N A a s e . All the a b o v e results w i t h s p i n a c h are essentially the s a m e as t h o s e of d e L o o z e a n d W a g n e r [1] w o r k i n g with Chenopodium. W e h a v e a p p r o a c h e d the q u e s t i o n of the physiological s i g n i f i c a n c e of these effects b y s u p p o s i n g that the R N A r e s p o n s i b l e m a y n o t e v e n be in the c h l o r o p l a s t s w i t h the g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e b u t o n l y a s s o c i a t e w i t h it u p o n cell a n d c h l o r o p l a s t b r e a k a g e . If this is the case, enz y m e s e x t r a c t e d f r o m c h l o r o p l a s t s p r e v i o u s l y isol a t e d i n t a c t f r o m the cell w o u l d n o t h a v e the R N A
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Fig. 1. Effect of N A D / N A D P on the elution profile of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Sephacryl S-300 columns. 1 ml of partially purified enzyme stored at - 18°C was thawed and applied to a Sephacryl S-300 column (1.3 × 39.5 cm) previously equilibrated with 50 mM Tris-HC1/5 mM EDTA/2 mM dithiothreitol/0.2 mM NAD ÷ (pH 7.5). Flow rate was 12 ml/h. 12 ml of eluate was rejected, then fractions of 1.1 ml were collected. Fractions 7-14 were collected, concentrated to 1 ml and rechromatographed on the same column but previously equilibrated with the above buffer containing 0.2 mM NADP +. • •, NAD+; or o - - -- - - o , NADP + in the chromatography buffer.
Fig. 2. Effect of high-molecular-weight fraction on the elution profile of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase. Enzyme was stored and chromatographed in an identical manner to Fig. 1, first in buffer containing 0.2 mM NADP + in place of NAD+. Fractions 8-12 were collected (4 ml) and called void-volume material. Enzyme activity eluted between fractions 15 and 23 and was collected as 8 ml. 4 ml of this was concentrated to 1 ml and rechromatographed on the same column equilibrated with buffer containing 0.2 ml NAD ® in place of NADP ÷. This elution profile is labelled o - - - - --o. 4 ml of enzyme remaining from the NADP ÷ chromatography was added back to 2 ml of the void volume material. This was then concentrated to 2 ml and 1 ml of this was rechromatographed on a column equilibrated with buffer containing 0.2 mM NAD ÷ in place of NADP ÷. This is the elution profile •
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189
present and would therefore exhibit altered aggregation phenomena. Fig. 3 shows an identical experiment to Fig. 1 except that enzyme has been extracted from previously isolated chloroplasts. It is clearly seen that even in the presence of N A D ÷ the bulk of the glyceraldehyde-3-phosphate dehydrogenase exists as a unit having a molecular weight of approx. 300000 and only a small part aggregates to the species of 600 000 or more. If, however, enzyme from chloroplasts is first mixed with the void volume fraction from a whole leaf preparation chromatographed on Sephacryl S-300 in the presence of N A D P +, then all activity aggregates in the presence of N A D ÷ to a species of molecular weight above 600000. The void volume fraction from the column of Fig. 3 run in the presence of N A D P ÷ has a ratio of absorbance 260 n m / 2 8 0 nm of 1.0, indicating a low concentration of RNA relative to protein. Fig. 4 shows that this void volume material fails to induce the NAD-dependent reassociation of enzyme prepared from whole leaves and previously disaggregated on a Sephacryl column run in the presence of N A D P ÷, whereas in an otherwise identical experiment void volume material from
whole leaf preparations induces NAD-dependent reassociation. The experiment entirely analogous to that of Fig. 4, but using dissociated glyceraldehyde-3phosphate dehydrogenase from previously isolated chloroplasts, gives identical elution profiles. Several groups have reported that NADP(H) which promotes the disaggregation of glyceraldeh y d e - 3 - p h o s p b a t e d e h y d r o g e n a s e activates NADP(H)-linked activity [2,3,9] by drastically reducing the K m for N A D P ( H ) [2]. The N A D ( H ) which promotes enzyme aggregation reverses this effect. Moreover non-Michaelis-Menten kinetic behaviour has been sometimes [2,5,9,13] but not always [14] reported. We have surmised that if aggregation phenomena are no longer seen in the absence of endogenous RNA, the associated changes in kinetic constants may also be absent. We have therefore begun the kinetic investigation of NADP-dependent glyceraldehyde-3-phosphate
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Fig. 3. Effect of N A D / N A D P on the elution profile of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase obtained from previously isolated chloroplasts. 1 ml of enzyme prepared from chloroplasts (see Materials and Methods) was chromatographed in an identical manner to Fig. 1. Enzyme chromatographed in buffer containing ([3 [3) 0.2 mM N A D +, (0-- ----0) 0.2 mM N A D P +,
was chromatographed on Sephacryl S-300 as in Fig. 2 in buffer containing 0.2 mM N A D P +, Fractions 8 - 1 2 were collected and called void volume material. Enzyme activity eluted between fractions 15 and 23 (8 ml) and was collected. An identical chromatography used 1 ml of enzyme from previously isolated chloroplasts and void volume material only was collected between fractions 8-12. 2 ml of enzyme from the first column were mixed with 1 ml of void volume material from either the first column (whole leaf preparation) or the second column (chloroplast preparation). Both samples were concentrated to 1 ml and chromatographed in buffer containing 0.2 mM N A D ÷. Elution profiles of enzyme with void volume material from (A A) whole leaf (m-- - - --m) isolated chloroplasts.
190
dehydrogenase from which has been removed the high-molecular-weight fraction containing RNA. We have used enzyme extracted from previously isolated chloroplasts or enzyme from whole leaf preparations which have been chromatographed on Sephacryl S-300 in the presence of N A D P + to remove the R N A fraction eluting with the void volume. Preincubation of enzyme at 25°C in the chro-
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matography buffer containing either 0.2 mM N A D + or 0.2 mM N A D P H leads to enzyme having identical activity in the normal reaction mixture when glyceraldehyde-3-phosphate dehydrogenase is added last to initiate the reaction. We have tested to see whether the failure to observe a difference in the activity between enzyme which has been presoaked in N A D * or N A D P H is due to activation by the N A D P H or A T P [3,9] of the assay mixture within the mixing time of a normal spectrophotometric experiment (approx. 10 s). Mixing time was reduced by using a rapid mixing and stopped flow spectrophotometer. Enzyme incubated in 0.2 mM N A D + and rapidly mixed to give standard assay conditions show no evidence of rate acceleration over the period 5 ms to 10 s. A previous study [2] has reported that the main activating effect of preincubation with N A D P ( H ) is to lower the K m for NADP(H). There is the possibility that our failure to observe any activating effects of N A D P ( H ) is due to the use during assay of near-saturating concentrations of N A D P H . Fig. 5 shows that with enzyme devoid of the high-molecular-weight RNA fraction, the K,1 for N A D P H is no different whether the enzyme is preincubated in N A D + or N A D P +. Typical Michaelis-Menten kinetics are observed in both cases.
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Fig. 5. Substrate saturation kinetics of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase previously equilibrated in (A) 0.2 m M N A D P +, (B) 0.2 m M N A D +. Glyceraldehyde-3-phosphate dehydrogenase from a whole leaf preparation was chromatographed on Sephacryl S-300 in the presnce of 0.2 mM N A D P + to remove void-volume material containing R N A and equlibrated against the same buffer as in Fig. 1 containing either 0.2 m M N A D + or 0,2 m M N A D P +. Samples were assayed in 100 m M Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM 2-mercaptoethanol, 8 m M MgSO 4, 2 mM ATP, 4.5 mM 3-phosphoglycerate, 1.6 units phosphoglycerate kinase, 0.015 units of glyceraldehyde-3-phosphate dehydrogenase and N A D P H as shown in a total volume of l ml. Line fitted by the method of Johansen and Lumry [15]. K,,, for N A D P H 25 # M (A) and 28 ,ttM (B).
The implication of several studies on N A D P dependent chloroplast glyceraldehyde-3-phosphate dehydrogenase has been that, in the presence of N A D +, a monomer of molecular weight 150000 aggregates to a tetramer of 600000 [2,7,16,17]. However, if an RNA-rich fraction which itself has a molecular weight of over 600000 induces glyceraldehyde-3-phosphate dehydrogenase in the presence of N A D + to elute from molecular exclusion chromatograms at a molecular weight of at least 600000, there seems little need to hypothesise a polymeric structure to account for the apparent increase in molecular weight. We are uncertain of the significance of the intermediate molecular weight profile of glyceraldehyde-3-phosphate dehydrogenase from isolated chloroplasts when first chromatographed in the
191
presence of NAD + (Fig. 3). It may be thought that an intermediate level of association is due to a low level of contamination of chloroplasts with extrachloroplastic RNA. This idea is supported by the fact that enzyme from previously isolated chloroplasts and chromatographed in the presence of N A D P + (which would free it from RNA eluting in the void volume) shows only the low molecular weight profile when rechromatographed in the presence of NAD +. Moreover, void volume material from chloroplasts preparations run on Sephacryl S-300 in the presence of NADP + has a slight tendency to promote NAD+-dependent reassociation of enzyme which has been freed from RNA (Fig. 4). In studies on enzyme activity we have not used enzyme concentrations as high as that used in chromatography experiments. There is still the possibility that at higher concentrations, enzyme free from nonchloroplast RNA does alter activity as the N A D / N A D P ratio changes. What is true is that these activity changes will be unrelated to association/dissociation phenomena, as Figs. 2 and 4 show none to exist in RNA-free enzyme preparations. The most extensive steady-state kinetic analysis of NADP-dependent chloroplast glyceraldehyde3-phosphate dehydrogenase published to date [14] shows no evidence of the non-Michaelis-Menten behaviour which would be revealed if NADP(H) activation was occurring. It is worth noting that this study [14] was accomplished with an enzyme which experienced, as an integral part of its purification, a molecular exclusion chromatography in the presence of N A D P +. This would have removed the crucial RNA fraction. Moreover, classical Michaelis-Menten behaviour was seen in another study which extracted enzyme from previously isolated chloroplasts [18]. Both of these studies are therefore consistent with the conclusion reached here that this enzyme behaves in a classical Michaelis-Menten fashion with no preactivation by NADP(H) required. In our hands NADP-dependent chloroplast glyceraldehyde-3-phosphate dehydrogenase has never shown non-Michaelis-Menten behaviour with
respect to NADPH, nor does N A D P H activate enzyme previously incubated in NAD +. In view of this study, however, and those of Cerff [14] and Speranza et al. [18], it may be useful to those who obtain such effects to investigate the role of contaminant RNA.
Acknowledgement We wish to thank for their assistance Dr. K. Brocklehurst and Dr. Francis Willenbrock of the Department of Biochemistry, St. Bartholomew's Hospital Medical School, London, in whose laboratory the stopped-flow experiments were carried out.
References 1 deLooze, S. and Wagner, E. (1983) Physiol. Plant. 57, 243-249 2 Pupillo, P. and Piccari, G.G. (1973) Arch. Biochem. Biophys. 154, 324-331 3 Pupillo, P. and Piccari, G.G. (1975) Eur. J. Biochem. 51, 475-482 4 Schwarz, Z., Maretzki, D. and Schonherr, J. (1976) Biochem. Physiol. Pflanzen 170, 37-50 5 Cerff, R. (1978) Eur. J. Biochem. 82, 45-53 6 Cerff, R. (1978) Plant Physiol. 61,369-372 7 Wara-Aswapati,O., Kemble, R.J. and Bradbeer, J.W. (1980) Plant Physiol. 66, 34-39 8 Pawlizki, K.H. and Latzko, E. (1974) FEBS Len. 42, 285-288 9 Wolosiuk, R.A. and Buchanan, B.B. (1976) J. Biol. Chem. 251, 6456 6461 10 Pupillo, P. and Faggiani, R. (1979) Arch. Biochem. Biophys. 194, 581592 11 Charles, S.A. and Halliwell, B. (1981) Cell Calcium 2, 211-224 12 Lilley, R.M.C., Fitzgerald, M.P., Rienits, K.G. and Walker, D.A. (1975) New Phytol. 75, 1-10 13 Ziegler, I., Marewa, A. and Schoepe, E. (1976) Phytochemistry 15, 1627-1632 14 Cerff, R. (1978) Phytochemistry 17, 2061-2067 15 Johansen, G, and Lumry, R. (1961) C.R. Trav. Lab. Carlsberg 32, 185-214 16 Ferri, G., Comerio, G., Iadarola, P., Zapponi, M.C. and Speranza, M.L. (1978) Biochim. Biophys. Acta 522, 19-31 17 Zapponi, M.C., Berni, R., ladarola, P. and Ferri, G. (1983) Biochim. Biophys. Acta 744, 260 264 18 Speranza, M.L., Zapponi, M.C. and Iadorola, P. (1982) ltal. J. Biochem. 31, 22-27