The Effect of High Hydrostatic Pressure on the Modulation of

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Vol. 266, No. 31, Issue of November 5, pp. 20913-20921,1991 Printed in U.S.A.

THEJOURNALOF

BIOLOGICAL CHEMISTRY 01991 by The American Society for Biochemistry and Molecular Biology, Inc.

The Effectof High Hydrostatic Pressure on the Modulation of Regulatory Enzymes from Spinach Chloroplasts* (Received for publication, May 8, 1991)

Gonzalo Prat-Gay$, Alejandro Paladini, Jr.6, Mariana Stein, and Ricardo A. Wolosiukn From the Instituto de Investigaciones Bioquimicas Fundacion Campomar and the Slnstituto de Ingenieria Geneticay Biologia Molecular, Buenos Aires, Argentina

High hydrostatic pressure enhanced the specific ac- In chloroplasts of higher plants, sunlight both provides tivity of regulatoryenzymes of the Benson-Calvin energy for the photosynthetic generation of ATP and NADPH cycle (fructose-l,6-bisphosphatase, glyceraldehyde-3- and controls regulatory enzymes (1-3). There is consensus P dehydrogenase,phosphoribulokinase)which are that the excitation of the photosynthetic electron transport modulated by the ferredoxin-thioredoxinsystem. High system triggers changes in stromal components which faciliactivity of chloroplast fructose- 1,6-bisphosphatasere- tate the conversion of enzymes to a form showing different kinetic properties. Changes induced by ions, metabolites, and quired dithiothreitol, fructose 1,6-bisphosphate, and Ca2+.At 100 bar the Ao.sfor fructose 1,6-bisphosphate the ferredoxin-thioredoxin system (ferredoxin, ferredoxin(0.3 mM) was lower than that at 1 bar (1.5 mM), thioredoxin reductase, thioredoxins) link light to enzyme regwhereas similar variations of pressure did not alter ulation. A common feature of these enzymes is that the rate the A0.6 for Ca2+(55 PM). The response of chloroplast of conversion from one form to another is slower than the glyceraldehyde-3-P dehydrogenaseexposed to 500 bar rate of catalysis (4,5). V, was a &fold increase in the NADP-linked activity; E n a c t i v e +E e m w conversely, the NAD-dependent activity remained unchanged. The concerted actionof high pressure andPi (or ATP), both activators of chloroplast glyceraldeSubstrates products V, > 5 h); similar to the pressuredrop from 1200 to 1 bar concentrations of Pi. Final volume: 0.2 ml. After 5 min, NADPstimulatory effect of high hydrostatic pressure on enzyme glyceraldehyde-3-P dehydrogenase activity was assayed in the soluactivity, changesinduced by decompression were slower than tion described under “Experimental Procedures.”

High Pressure Action on Chloroplast Enzymes

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the Aoa for primary modulators; on the contrary, it caused the loss of enzyme activity. When high pressure was exerted on the catalytic process, the rate of NADP reduction was consistently low (not shown). At thisstage of the investigation, two alternative explanations accounted for the lack of NADP-linked activity; high hydrostatic pressure either inhibited catalysis or inactivated chloroplast glyceraldehyde-3-Pdehydrogenase in concerted action with metabolites present in the assay solution. Phosphoribulokinhe-Phosphoribulokinase, an enzyme characteristic of autotrophic organisms, catalyzes the phosphorylation at the C-1 in ribulose 5-P, yielding ribulose 1,5bisphosphate (the acceptor for CO, in the Benson-Calvin cycle) (47),

phase of the Benson-Calvin cycle. Ribulose 1,5-P2 + CO, + 2,3-phosphoglycerate M%2+

Paradoxically the catalytic activity of this, the most abundant protein of the world, is low; however, prior exposure to both COZ and M e potentiates the capacity for fixing COz (48,49). Clearly, the rate of activation of ribulose 1,5-bisphosphate carboxylase is slower than catalysis, i.e. it is a hysteretic enzyme. This feature facilitates the interpretationof the dual action that some chloroplast components exert on the activity; e.g. like found for chloroplast fructose-1,6-bisphosphatase, Ca2+enhances the conversion of inadive ribulose 1,5-bisphosphate carboxylase to an active form, whereas it inhibits the catalyticstep (7, 50). In contrast to other regulatory enzymes of the Benson-Calvin cycle, ribulose-1,5-bisphosribose 5-P ribulose 5-Pribulose 1,5-P2 phate carboxylase is not controlled by the ferredoxin-thioredoxin system (51). Therefore, on the basis of its chloroplast ADP ATP localization, the hysteretic behavior, and the lack of thiorewhere @ is phosphoribose isomerase and @ is phosphori- doxin stimulation, we chose ribulose-1,5-bisphosphatecarboxylase as the counterpart (control) for the analysis of the bulokinase. In chloroplasts of higher plants, the enzyme is controlled action of nonphysiological modulators. Withthis goal in mind we determined that chaotropic by thioredoxin, reduced either chemically with dithiothreitol or photochemically with the ferredoxin-thioredoxin system anions were potentinhibitors of ribulose-l,5-bisphosphate (31). Alternatively, the catalytic capacity is stimulated by a carboxylase activation, whereas the effect of kosmotropic salts thioland an organic solvent; the lower the octanol/water was less pronounced 37 mM sodium-trichloroacetate and 0.6 partition coefficient of the cosolvent, the less its concentration M Na2S04lowered the specific activity to half of the maximum, respectively. On the other hand, 10% (v/v) 2-propanol for maximal stimulation (13, 18). Inthe presence of 5 mM of dithiothreitol, the specific did not change the specific activity of ribulose-1,5-bisphosactivity of phosphoribulokinase increased 10-fold by raising phate carboxylase; higher concentrations of the cosolvent the pressure to 1500 bar and remained high even at 2000 bar precipitated the enzyme (55). To establish the effect of high hydrostatic pressure, we (the highest compression delivered by our instrument) (Fig. carboxylase in a solution 7). In this regard, high pressure acted as the secondary mod- incubated ribulose-1,5-bisphosphate ulator, because the presence of areductant (thiol-bearing containing both HCO, and M e at different pressures, and compound) was strictly required for the stimulation of kinase subsequently we estimated the ribulose 1,5-bisphosphate-dependent fixation of I4CO2at atmospheric pressure. As depicted activity. As shown in Scheme, both the substrate(ribulose 5-P) and in Fig. 8, the specific activity of the enzyme decayed whenthe the product (ADP) were enzymatically generated and esti- pressure was raised to 2000 bar. Although basal activity (at 1 mated, respectively. Investigation of high pressure on the bar) was higher when the concentration of HCO; and M$+ catalytic process of phosphoribulokinase was rendered diffi- was increased to 8 and 20 mM, respectively, such incorporation cult, because the inhibitory effect of high pressure on the to the solution did not prevent pressure-mediated inactivation. These results showed that, in contrast to thioredoxinactivity of auxiliary enzymes made the analysis less reliable. NADP-glycRibulose 1,5-BisphosphuteCarboxylase-The other enzyme activated enzymes (fructose-l,6-bisphosphatase, characteristic of autotrophic organisms is ribulose 1,5-bis- eraldehyde-3-P dehydrogenase, phosphoribulokinase), the phosphate carboxylase which catalyzes the carboxylative

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1000

2000

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Pressure (bar1

FIG. 7. Effect of high hydrostatic pressure on the activation of phosphoribulokinase. Phosphoribulokinase (40 pg) was incubated at 23 “C in 0.1 M Tris-HC1 buffer (pH 7.9) containing 5 mM dithiothreitol. Finalvolume: 0.2 ml. After 5 min atthe indicated pressure, an aliquot was withdrawn and injected into the solution for the assay of phosphoribulokinase activity described under “Experimental Procedures.”

FIG. 8. Inactivation of ribulose-l,5-bisphosphatecarboxylase by high hydrostatic pressure. Ribulose-1,5-bisphosphate carboxylase (280 pg) was incubated at 25 ”C in a solution containing the following (in micromoles); Bicine-KOH buffer (pH 8.0), 10; dithiothreitol, 5; and, as indicated, NaHCOa and MgC12. After 10 min under varying pressures, aliquots werewithdrawn and assayed for ribulose-1,5-bisphosphatecarboxylase activity, as describedunder “Experimental Procedures.”

High Pressure Action on Chloroplast Enzymes specific activity of a thioredoxin-insensitive regulatory enzyme wasnot stimulatedby nonphysiological modulators. On the contrary, the conversion of inactive ribulose-1,5-bisphosphate carboxylase to an active form was either inhibited by chaotropic anions andhigh hydrostatic pressure or unaffected by cosolvents. DISCUSSION

The pressure-mediated activation of chloroplast enzymes may not normally beof physiological importance because most photosynthetic organisms thrive in environments with pressure lower than 5 bar.However, this physical perturbant is of considerable importance in investigating the structural requirements for the thioredoxin-mediated activation of chloroplast enzymes. At constant temperature, the pressure dependence of reaction velocity is due entirely to theactivation volume of the reaction (23),

where k is the rate constant, P is pressure, T is temperature, AVffis the activation volume, and R, gas constant (0.083 liter. bar. K" .mol"). Since we could not separate experimentally all different stages involved in the conversion of the inactive enzyme to the active form (modulator binding, reduction, conformational change), we considered the activation process as one step. As a consequence, theapparent AV' would indicate the variation of volume between the final (active) and initial (inactive) states of the enzyme, irrespective of other substrates in such transition. The positive slope, obtained in plots of specific activity uersus pressure, indicated that a decrease in volume accompanies the formation of active states inregulatory enzymes of chloroplasts; conversely, high pressure inactivates other enzymes, either from photosynthetic systems or from heterotrophic organisms. Using the above equation for our kinetic results, we calculated negative activation volumes for the following enzymes of the BensonCalvin cycle: glyceraldehyde-3-P dehydrogenase, -56 mlmol"; phosphoribulokinase, -36 ml. mol"; and fructose-1,6bisphosphatase: -250 ml. mol". On the other hand, a positive activation volumewas found for ribulose-1,5-bisphosphate carboxylase (+34 ml.mol"). In the analysis of kinetic results, we compared enzymes of different structures, Le. homo- and heteropolymers. Chloroplast enzymes composed of identical (fructose-1,6-bisphosphatase, phosphoribulokinase) or different (glyceraldehyde-3-P-dehydrogenase) subunits were stimulated by high pressure. In contrast, enzymes constituted by similar (mammalian fructose-l,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase) or dissimilar (ribulose-1,5bisphosphate carboxylase) subunits were inactivated by high pressure (40, 45, 48).As Table I11 illustrates, the high pressure-mediated enhancement of enzyme activity correlated with thioredoxin-dependent activationrather than with a particular constitution of the enzyme. Thus, we concluded that the stimulatory action of pressure on activity depended on intrinsic characteristics of the enzyme, and it was not related to thesubunit composition. Moreover, the parallelism between the thioredoxin- and high pressure-mediated activation was independent from the reaction that was subsequently catalyzed; both the physiological and nonph~~siological modulators enhanced the specific activity of a phosphatase, a dehydrogenase, and a kinase. In our studies, two aspects of chloroplast enzymes were different to published results on the effects of high pressure. Penniston (52) proposed that high pressure stimulates the activity of monomeric enzymes and inhibits the catalytic

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TABLE111 Enzyme structure and the activation by high pressure and thioredoxin Activation by Enzyme, structureof the catalytically active High Thioredoxin form pressure Chloroplast fructose-1,6-bisphosphatase: homotetramer, subunit molecular weight 40,000 (39) Chloroplast glyceraldehyde-3-P dehydrogenase: 8 subunits of molecular weight 37,000, and 8 subunits of molecular weight 43,000 (43) Chloroplast phosphoribulokinase: homodimer, subunit molecular weight 44,000 (47) Chloroplast ribulose-1,5 bisphosphate carboxylase: 8 subunits of molecular weight 56,000and 8 subunits of molecular weight 14,000 (48) Mammalian fructose-1,6-bisphosphatase: homotetramer, subunit molecular weight 35,000 (40) Yeast glyceraldehyde-3-P dehydrogenase: homotetramer, subunit molecular weight 36,000 (45)

Yes

Yes

Yes

Yes

Yes

Yes

NO

No

No

No

No

No

capacity of oligomeric enzymes. However, the experimental separation of enzyme modification from the catalytic reaction allowed us to show that, in the presence of specific modulators, high hydrostatic pressure enhanced the activity of chloroplast (oligomeric) enzymes, irrespective of the polymerization degree (Table 111). Thus,the effect of pressure on activity depended not only on the enzyme, but it required a precise definition of the process under consideration. Second, the modification of protein structureby high hydrostatic pressure was not aquasi-reversible process, because neither chloroplast fructose-l,6-bisphosphatasenor chloroplast glyceraldehyde3-P dehydrogenase returned to initial state (low specific activity) after decompression, uiz. pressure-mediated enhancement of activity was an irreversible process (24). However, in our considerations, we cannot rule out that the stability of active chloroplast fructose-l,6-bisphosphatasewas caused by the additional action of the reductant and modulators. On the basis of specific requirements for high pressure mediated stimulation of enzyme activity, it was reasonable to assume that only selected regions of oligomeric proteins changed its size under pressure. Therefore, the resultant AVff represents a scalar average of multiple changes in protein solvation. Jaenicke compiled AVffassociated with model reactionsrelevant to biochemistry (cf. TableI in Ref. 25). Hence, the sign and value of the experimental AVffallowed to estimate the interactions that play a key role in the activation process. On this basis, the AVn for chloroplast glyceraldehyde3-P dehydrogenase and phosphoribulokinase suggested the preeminence of hydrophobic hydration over other processes (e.g ion pairformation). Although the sign was similar, modifications around small groups could not account for the AVffof chloroplast fructose-l,6-bisphosphatase, because high negative values imply large variations in the structure. Evidence that theactivation process caused large changes in the surface of chloroplast fructose-1,6-bisphosphatasewas the finding that the active and inactive forms interacted differentially with micelles of detergents? Numerous arguments converge in supporting the proposal that intramolecular hydrophobic interactions of chloroplast enzymes are modified in the activation process. The increase of enzyme activity by

* G. Prat-Gay, unpublished data.

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High Pressure Action onChloroplast Enzymes

organic solvents correlated inversely with Poctanollwater; the ylase diminished by chaotropic anions and remained unalmore hydrophobic the cosolvent, the less its concentration for tered in the presence of cosolvents. The comparison of several maximalstimulation (8, 12, 13). On theotherhand,the enzymes revealed that the stimulatoryeffect of physical and enhancement of the specific activity by neutral salts was chemical modulators was observed in those regulated by the described by the lyotropic (Hofmeister) series; the concentra- ferredoxin-thioredoxinsystem (fructose-1,6-bisphosphatase, tion of chaotropic anions for maximal stimulation was lower NADP-glyceraldehyde-3-P dehydrogenase, phosphoribulokithan that of kosmotropes (14, 15, 17). Studies in other labo- nase). Conversely, either from chloroplast (ribulose-1,5-bisratories (19,22, 25) showed that chemical and physical mod- phosphate carboxylase) or from nonphotosynthetic sources ulators used by us altered the characteristics of protein hy- (mammalian fructose-l,6-bisphosphatase,yeast glyceraldedration whereby changing intramolecular interactions. In thishyde-3-P-dehydrogenase),other oligomeric enzymes did not analysis,it is important to bear in mind that cosolvents, increase their catalytic capacityby nonphysiological modulators. However, we envisage that the stimulationof activity by chaotropic anions, and high pressure did not participate in redox mechanisms. Therefore, our results on the action of nonphysiological modulators would appear in otherenzymes, catalysis. nonphysiological modulatorsonchloroplast regulatory en- if perturbants were tested on reactions other than Although the primary structure of thioredoxins showed a zymes indicated that themodification of hydrophobic interredox activecenter(-Trp-Cys-Gly-Proactions is an important mechanism in the activationof thio- constancyinthe Cys-), their action on chloroplastenzymes showed large varredoxin-regulated enzymes. Although the response of chloroplast regulatory enzymes to iations (53, 54). The activation of chloroplast enzymes with modulators that do not take part in redox reactions suggests high pressure had similar characteristics, an important feature should be emphasized; the presence of a reductant (di- that thioredoxin playsa structural role in enzyme regulation. thiothreitol) was strictly required for the activation of fruc- The lack of reductive mechanisms in other thioredoxin-metose-1,6-bisphosphatase and phosphoribulokinase, whereas itdiated processes, the promotionof T 7 phage DNA polymerase was not essential to stimulate NADP-glyceraldehyde-3-P de-activity and the replicationof filamentous phages, speaks in favor of that view (10,ll). On this basis, it appears that other hydrogenaseactivity.Moreover, both fructose1,6-bisphosphate and Ca2+were necessary for the stimulation of chloro- domains of thioredoxin participate in modifying intramolecplast fructose-1,6-bisphosphatase;high pressure, acting as a ular interactionsof key enzymes of chloroplasts. secondary modulator, lower the Ao.&for fructose 1,6-bisphosAcknowledgments-We are indebted to Drs. G . Weber and B. B. phate, whereas it did not change the A0.5 Ca2+. for Clearly, in Buchanan for helpful comments. the enhancement of the specific activity of fructose-1,6-bisphosphatase and phosphoribulokinase, high pressure was kiREFERENCES netically indistinguishable from thioredoxin, cosolvents, and 1. Woodrow, I. E., and Berry, J. A. (1988) Annu. Reu. Plant Physiol. chaotropic anions; namely, both physiological and nonphysi39,533-594 ological modulatorsacted as secondarymodulatorsinthe 2. Buchanan, B. B., Wolosiuk, R. A., and Schurmann, P. (1979) Trends Biochem. Sci. 4,93-96 concerted hysteresis phenomena ( 5 ) . At variance with fruc3. Buchanan, B. B. (1980) Annu. Reu. Plant Physiol. 31,341-374 tose-1,6-bisphosphataseandphosphoribulokinase, chloro4. Wolosiuk, R. A., andHertig, C. M. (1983),in Thioredoxins, plast glyceraldehyde-3-P dehydrogenase was the only thioreStructure and Functions (Gadal, P., ed) pp. 167-173, Editions doxin-controlled enzyme in which cosolvents, chaotropic andu CNRS, Paris ions, and high pressure acted as both primary and secondary 5. Frieden, C. (1970) J. Biol. Chem. 245, 5788-5799 modulators (8, 17). The stimulation obtainedwith high pres6. Wolosiuk, R. A., Perelmuter, M. E., and Chehebar, C. (1980) FEBS Lett. 109,289-293 sure, acting as primary modulator, was similar to thatcaused 7. Hertig, C., and Wolosiuk,R. A. (1980) Biochem. Biophys. Res. by either thioredoxin,cosolvents, or chaotropic anions.HowCommun. 97,325-333 ever, the behavior of high pressure, as secondary modulator, 8. Wolosiuk, R. A., Hertig, C. M., and Busconi, L. (1986) Arch. was opposite to biochemical and chemical modulators. ThioBiochem. Biophys. 246, 1-8 redoxin,organic solvents, and chaotropic anions enhanced 9. Holmgren, A. (1985) Annu. Reu. Biochem. 54, 237-271 the action of metabolites like Pi, ATP, NADPH, i.e. as sec- 10. Tabor, S., Huber, H. E., and Richardson, C. C. (1986) in Thioredoxin and Glutaredoxin Systems:Structure and Function ondary modulators, lowered the A0.5of primary modulators. (Holmgren, A., ed) pp. 285-300, Raven Press, New York Conversely, high pressure inhibited the stimulatory action of 11. Model, P., and Russell, M. (1986) in Thioredoxin and Glutarethese intermediatesof the Benson-Calvincycle; paradoxically, donin Systems: Structure and Function (Holmgren, A., ed) pp. each modulator separately enhanced the specific activity of 323-329, Raven Press, New York glyceraldehyde-3-P dehydrogenase, but their concerted action 12. Corley, E., and Wolosiuk, R. A. (1985) J. Biol. Chem. 260,39783983 caused the inactivation of the enzyme. This particular result constituted the first evidence that thioredoxin and high hy- 13. Wolosiuk, R. A,, Corley, E., Crawford, N. A,, and Buchanan, B. B. (1985) FEBS Lett. 189,212-216 drostatic pressure differed in some unknown feature, even 14. Crawford, N. A,, Yee, B. C., Hutchenson, S. W., Wolosiuk, R. A., though their action on chloroplast enzymes was similar in and Buchanan, B. B. (1986) Arch. Biochem. Biophys. 244, 1several aspects. An important characteristic in the activation 15 of chloroplast glyceraldehyde-3-P dehydrogenase was the ab- 15. Stein, M., and Wolosiuk, R. A. (1987) J. Bid. Chem. 262, 1617116179 sence of a reductant to elicit the stimulatory action of non16. Stein, M., Lazaro, J. J., and Wolosiuk, R. A. (1989) Eur. J . physiological modulators. T h e analysis of such differences Biochem. 185, 425-431 with fructose-1,6-bisphosphataseandphosphoribulokinase 17. Wolosiuk, R. A,, and St.ein, M. (1990) Arch. Biochern. Biophys. raises a question for future experiments: dononphysiological 279, 70-77 18. Lea, A., Hansch, C., and Elkins, D. (1971) Chem. Reu. 71, 525modulators mediate in the modification of enzyme confor616 mation or in reshuffling -S-S-/-SH couples? K. D.. and Washabauah. 19. Collins. . . - . M. W. (1985) 9. Reu. Bi0ph.y~. Incontrastto regulatoryenzymes, ribulose-l,5-bisphos18,323-422 phate carboxylase was inactivated by high pressure in the 20. Low, P. S., and Somero, G. N. (1975) Proc. Natl. Acad. Sci. presenceandinabsence of modulators (CO,, Mg2+)(51). U. S. A. 72, 3305-3309 Moreover, the activity of ribulose-1,5-bisphosphatecarbox- 21. Nemethy, G., Peer, W., and Scheraga, H. A. (1981) Annu. Reu.

High on Pressure Action Biophys. Bioeng. 10,459-497 22. Zaks, A., and Klibanov, A.M. (1988) J. Biol. Chem. 263,80178021 23. Morild, E. (1981) Adu. Protein Chem. 3 4 , 93-166 24. Weber, G., and Drickamer, H. G . (1983) Q.Reu. Biophys. 16,89112 25. Jaenicke, R. (1983) D.Naturwiss. 70,332-341 26. Seifert, T., Bartholmes, P., and Jaenicke, R. (1984) FEBS Lett. 173,381-384 27. Kornblatt, M. J., and Hui Bon Hoa, G. (1987) Arch. Biochem. Biophys. 252, 277-283 28. Hertig, C. M., and Wolosiuk, R. A. (1983) J. Biol. Chem. 258, 984-989 29. Hall, N. P., and Tolbert, N.E. (1978) FEBS Lett. 96, 167-169 30. Wolosiuk, R. A., andBuchanan, B. B. (1976) J . Biol. Chem. 2 5 1 , 6456-6461 31. Wolosiuk, R. A., and Buchanan, B. B. (1978) Arch. Biochem. Biophy~.189,97-101 32. Geller, A. M., and Byrne, W. L. (1975) Methods Enzymol. 43, 363-368 33. Paladini, A. A., Jr., and Weber, G. (1981) Biochemistry 20,25872593 34. Neuman, R.C., Kauzmann, W., and Zipp, A. (1973) J . Phys. Chem. 77,2687-2691 35. Chen, P. S., Toribara, T. Y., and Warner, H. (1956) Anal. Chem. 28,1756-1758 36. Wolosiuk, R. A., Crawford, N. A., and Buchanan, B. B. (1977) FEBS Lett. 81,253-258 37. Chehebar, C., and Wolosiuk, R. A. (1980) Biochim. Biophys. Acta 613,429-438

Chloroplast Enzymes

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38. Greaney, G. S., and Somero, G. N. (1979)Biochemistry 18,53225332 39. Raines, C. A., Lloyd, J. C., Longstaff, M., Bradley, D., and Dyer, T. (1988) Nucleic Acids Res. 16, 7931-7942 40. Horecker, B. L., Melloni, E., andPontremoli, S. (1975) Adu. Enzymol. 42,193-226 41. Balny, C., and Hooper, A. B. (1988) Eur. J.Biochem. 176, 273279 42. King, L., and Weber, G. (1986) Biochemistry 25,3632-3637 43. Iadarola, P., Bonferoni, C., Ferri, G., Stoppini, M., and Zapponi, M. C. (1986) J. Chromatogr. 359,423-432 44. Wolosiuk, R. A., and Buchanan, B. B. (1978) Plant Physiol. 61, 669-671 45. Jaenicke, R., Schmid, D., and Knof, S. (1968) Biochemistry 7, 919-926 46. Ruan, K., and Weber, G. (1988) Biochemistry 27,3295-3301 47. Porter, M. A., Milanez, S., Stringer, C. D., and Hartman, F. (1986)Arch. Biochem. Biophys. 245, 14-23 48. Miziorko, H. M., and Lorimer, G. H. (1983) Annu. Reu. Biochem. 52,507-535 49. Lorimer, G. H., Badger, M. R., and Andrews, T. J. (1976) Anal. Biochem. 78, 66-75 50. Barcena, J. A. (1983) Biochem. Intern. 7,755-760 51. Tenaud, M., and Jacquot, J. P. (1987) J. Plant. Physiol. 130, 315-326 52. Penniston, J. T. (1971) Arch. Biochem. Biophys. 1 4 2 , 322-332 53. Maeda, K., Tsugita, A., Dalzoppo, D., Vilbois, F., and Schurmann, P. (1986) Eur. J. Biochem. 154, 197-203 54. Schurmann, P., Roux, J., and Salvi, L. (1985) Physiol. Veg. 23, 813-818 55. Prat-Gay, G. (1992) J. Biol. Chem., in press