Journal o f Protein Chemistry, VoL 10, No. 3, 1991

Protection and Enhancement of Ribulose 1,5 Bisphosphate Carboxylase Activity by Exogenous Proteins Lola Pefiarrubia l'z and Joaquin Moreno I

Received January 28, 1991

When assayed in vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase is both enhanced and protected from spontaneous decay by exogenous proteins such as hemoglobin, serum albumin, and aldolase. Other proteins and amino acids tested are either ineffective (lysozyme, ferritin, lysine, and cysteine) or afford only partial protection (catalase, glycine, and phenylalanine). Protective proteins do not bind to, or exchange disulfides with, ribulose 1,5 bisphosphate carboxylase/oxygenase. Since their effect can be mimicked by reductively treated detergents such as Triton X-100, it appears that proteins protect from decay by quenching the spontaneous oxidative degradation and inhibiting surface adsorption which could lead to enzyme unfolding. Release of adsorbed molecules from the container surface is likely to be the cause of carboxylase activity enhancement. KEY WORDS: Ribulose 1,5 bisphosphate carboxylase/oxygenase; enzyme protection; enzyme inactiviation; protein oxidation; protein structural stability (Citrus).

1. INTRODUCTION 3

fallover is due to an inhibitory by-product arisen from the catalytic transformation of the phosphorylated sugar (Edmondson et al., 1990). We have postulated that irreversible inactivation occurs through partial unfolding of the enzyme and stabilization of the scrambled structure by oxidation of some cysteine residues (Pefiarrubia and Moreno, 1987). We reported, however, that the addition of bovine serum albumin to RuP2 carboxylase/oxygenase solutions prevents spontaneous inactivation by a mechanism different from providing protection against proteinases. Moreover, the presence of serum albumin results also in a 10-15% increase of the RuP2 carboxylase activity (Pefiarrubia and Moreno, 1987). Since the nature of the interaction between serum albumin and RuP2 carboxylase/oxygenase is not known, we have attempted to explain the dual effect of albumin (which can be extended to some other proteins, as shown below) by looking for plausible mechanisms of interaction that could result in RuP2 carboxylase activation and/or protection. This may help to better understand the spontaneous inactivation of RuP2 carboxylase/oxygenase, as well as to prevent decay of enzyme preparations.

The crucial photosynthetic and photorespiratory enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase (RuP2 carboxylase/oxygenase, also known as Rubisco) has been reported to undergo spontaneous inactivation in solution (Hall et al., 1981; McCurry et al., 1982; Makino et al., 1983; Servaites, 1985; Gezelius and Widell, 1986; Pefiarrubia and Moreno, 1987; Heuer and Portis, 1990). This process (called irreversible inactivation in the literature) is different from the reversible loss of the Mg 2+-carbamate complex at the Lys2°~ residue, which is needed for a catalytically competent form of the protein (Miziorko and Lorimer, 1983).. It differs also from the decline of activity observed during catalysis (termed "fallover") in that irreversible inactivation takes place in the absence of the substrate ribulose 1,5-bisphosphate, while ~Departament de Bioqulmica i Biologia Molecular, Facultats de Ciencies, Universitat de Valencia, Dr. Moliner 50, Burjassot 46100 (Valencia) Spain. 2 Present address: Plant Biology Department, 111 Gene and Plant Biology Building, University of California, Berkeley, California 94720. 3 Abbreviation used: RuP2, ribulose 1,5 bisphosphate.

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Pefiarrubia and Moreno

2. MATERIALS AND METHODS

The purification of RuP2 carboxylase/oxygenase from leaves of the orange tree (Citrus sinensis L. Osbeck cv Washington Navel) has been described previously (Pefiarrubia and Moreno, 1988). All the experiments were performed with more than 95% pure RuP2 carboxylase/oxygenase as estimated by densitometry after SDS gel electrophoresis and protein staining (Conejero and Semancik, 1977). Carboxylase activity was determined according to Lorimer et al. (1977), with some modifications that optimized the procedure for the citrus enzyme (Pefiarrubia and Moreno, 1987). Exogenous proteins were obtained from Sigma except for catalase, aldolase, and egg albumin from Boehringer. 3. RESULTS The effect of several proteins and other amino and amido compounds on the initial activity and inactivation rate of RuP2 carboxylase/oxygenase is shown in Table I. Some proteins (like serum albumin, hemoglobin, thyroglobulin, egg albumin, and aldolase) were able to enhance the initial carboxylase activity, while others (ferritin, lysozyme) had no significant Table I. Effect of Different Proteins, Aminoacids, and Urea on

RuP2 Carboxylase Activity and Stabilitya % activity Compound added None Serum albumin Hemoglobin Lysozyme Ferritin Catalase Aldolase Thyroglobulin Egg albumin Glycine Cysteine Lysine Phenylalanine Urea

0 hr

26 hr

100 l 18 134 104 101 106 114 119 116 107 103 104 102 96

9 114 129 18 17 41 109 19 8 58 17 16 53 11

a Purified RuP2 carboxylase/oxygenase was dissolved (0.2 mg/ml) in 100 mM Tris HCI, 20 mM MgCI2, 10 mM NaHCO3 (pH 7.5). Compounds listed below were added at 1 mg/ml (final concentration) and carboxylase activity was determined initially (time 0) and after 26 hr of incubation at 30°C. Results are given as percentage of the initial activity without additions [1170 nmol CO2 fixed ( m i n m g RuP2 carboxylase/oxygenase)-1]. Values are means of two independent experiments, each of them with duplicate activity determination.

effect. Amino acids and urea were not particularly effective as activating agents. After 26 hr of incubation, some of the activity-enhancing proteins (serum albumin, hemoglobin, aldolase, and to a lesser extent catalase) also showed a protective effect (Table I). All other proteins failed to protect the RuP2 carboxylase/ oxygenase from inactivation. Among the amino acids tested, glycine and phenylalanine provided partial protection while urea was ineffective (Table I). These results indicate that both activation and protection of the RuP2 carboxylase/oxygenase are properties displayed only by specific proteins or amino acids. Hence, the cause for these effects should be sought in certain differential characteristics among them. Since the activating and protecting properties are not strictly correlated, they might be the result of different physical interactions. A trivial reason for activity enhancement could be the presence of carbonic anhydrase as a contaminant in the commercial preparations of the activating proteins. Carbonic anhydrase could increase the measured carboxylase activity if the availability of carbon dioxide from bicarbonate were limiting the reaction rate. This possibility was discarded because the difference in activity between presence or absence of the proteins persisted after the addition of an excess of carbonic anhydrase to the assay medium (data not shown). In order to examine the possibility that the structure of RuP2 carboxylase/oxygenase could be stabilized through a direct yet unspecific association with the exogenous proteins, a binding assay was performed according to Ackers (Ackers, 1975), using hemoglobin as protective protein. No evidence of binding between hemoglobin and RuP2 carboxylase/oxygenase was found (data not shown). The sensibility of the method excluded an association constant higher than 0.05/.tM- 1. Since the activity lost by spontaneous inactivation is partially recovered through treatment with thiol compounds (Hall et al., 1981; McCurry et al., 1982; Pefiarrubia and Moreno, 1987), it is likely that oxidation of cysteine residues is involved in the carboxylase decay. Determination of free cysteines indicated indeed a drop of four to five (out of 92 cysteines per molecule) on the number of titrable sulfhydrils of RuP2 carboxylase/oxygenase after spontaneous decay to 50% of its initial activity (Pefiarrubia and Moreno, 1990). Hence, it seems plausible that spontaneous inactivation of RuP2 carboxylase/oxygenase could be partially due to oxidation of some critical residues.

Ribulose 1,5 Bisphosphate Carboxylase Activity Table II. Comparison of the Effect of Nonmodified vs. Iodoaceta-

mide-Treated Proteins on RuP2 Carboxylase Activity and Stabilitya % activity Protein None Serum albumin Alkyl-serum albumin Hemoglobin Alkyl-hemoglobin Lysozyme Alkyl-lysozyme

0 hr

33 hr

100 118 119 134 134 102 103

20 106 88 115 119 30 8

a Iodoacetamide-treated proteins (Creigton, 1980) were added at 1 mg/ml (final concentration) to RuP2 carboxylase/oxygenase solutions prepared as in Table I. Carboxylase activity was determined initially (0 hr) and after 33 hr of incubation at 30°C.

Molecular oxygen could act as an oxidant, the reaction being catalyzed by traces of metallic ions in solution (Jocelyn, 1972). According to that, protection might result of the disulfide exchange with the free thiol groups of the exogenous proteins [intramolecular exchange has been shown to happen in the case of serum albumin (Jocelyn, 1972)]. This possibility was investigated blocking the sulfhydryl groups of serum albumin, hemoglobin, and lysozyme with iodoacetamide. The alkylated proteins showed the same protecting and enhancing properties as the untreated ones (Table II); therefore, the disulfide exchange explanation was also discarded. However, proteins could protect the RuP2 carboxylase/oxygenase against oxidative inactivation by other means not involving disulfide exchange (e.g., they could act as a sink for oxidative agents). To check this possibility, oxidative inactivation of RuP2 carboxylase/oxygenase was induced with CuSO4 (Tenaud and Jacquot, 1987), both in the presence and absence of a protective protein (hemoglobin). Results in Table III show that hemoglobin indeed provided some protection when compared with the faster decay observed in its absence. It is most likely that under the strong oxidative

289 conditions obtained when copper ions are added to the medium, the protective effect of hemoglobin is not sufficient to prevent partial decay. The dissimilar protective properties of different proteins may correlate with the exposure of easily oxidizable residues that could quench the mildly oxidant medium. However, such correlation is not observed among the amino acids (where cysteine has no effect, while glycine and phenylalanine afford some protection) (Table I). Furthermore, this effect does not explain the initial enhancement of activity caused by hemoglobin and other proteins. Another compatible explanation for activity decay is the adsorption of the enzyme at interfaces. Proteins are known to unfold and inactivate when bound to the container inner surface (Macritchie, 1978). RuP2 carboxylase/oxygenase has been shown indeed to absorb to surfaces when subjected to mild shear stress (Pefiarrubia and Moreno, t987). The possibility of a surface effect was examined adding Triton X-100 [a detergent that prevents protein adsorption (Suelter and DeLuca, 1983)] to the incubation medium. The presence of 0.1 mM Triton X100 resulted in a consistent increase (10-15%) of the initial activity, but the activity decreased afterward at a rate similar to the control (Fig. 1). However, when

120 &

80 >

TaMe Ill. Effect of Copper Sulfate and Hemoglobin on RuP2 0

Carboxylase Activity and Stability" Compound added None Hemoglobin Copper sulfate Copper sulfate + hemoglobin

0 min 100 131 96 128

% activity 80 min 165 min 74 124 43 85

39 123 13 54

°Hemoglobin (1 mg/ml) and/or copper sulfate (50/m a) were added to RuP2 carboxylase/oxygenase solutions (0.2 mg/ml), prepared as in Table I. Carboxylase activity was assayed at time 0, 80, and 165 rain.

I 0

i 8 time

i 16

~

{h)

Fig. 1. RuP2 carboxylase/oxygenase (0.3 mg/ml) in i00 mM TrisHC1, 10 mM MgCI2, 10 mM NaHCO3 (pH 7.5) ( 0 ) , with 0.1 mM Triton X- 100 ( A ), 10 mM 2-mercaptoethanol ( Q ), or 0.1 mM Triton X-100 and 10 mM 2-mercaptoethanol (&). In the latter case, Triton X-100 and 2-mercaptoethanol were previously mixed at a 10-fold concentration 30 min before addition to the enzyme. Points values are means of two independent experiments, each of them with duplicate activity determination.

290

Triton X-100 was pretreated with 2-mercaptoethanol in order to reduce the oxidizing impurities that are usually present in commercial preparations of this detergent (Ashani and Catravas, 1980), it showed also protective properties (Fig. 1). 2-mercaptoethanol alone did not afford protection but rather inhibited slightly the carboxylase activity as previous reported (Pefiarrubia and Moreno, 1987). On the other hand, the use of siliconized vials prevented activity decay to a similar degree than did Triton X-100 (data not shown). 4. DISCUSSION The above results suggest that RuP2 carboxylase/ oxygenase molecules stick to the inner surface of the container and that some protective agents (proteins, amino acids, or detergents) could act as surface adsorption inhibitors by competing for the adsorption sites. The protective effect may be explained assuming that spontaneous inactivation is mediated or catalyzed by surface adsorption. It is likely that the strain induced by the interaction with the surface could facilitate the partial unfolding of the enzyme (Macritchie, 1978). In this case, protective activity of proteins would correlate with the ability of binding to adsorption sites. On the other hand, adsorbed molecules, even if not unfolded, would be less active due to lower accessibility of the substrates to the catalytic site. Therefore, most of the substances that inhibit surface adsorption should both increase and preserve the activity as observed. The data presented above, together with evidence reported elsewhere (Pefiarrubia and Moreno, 1987), suggest that spontaneous inactivation of the RuP2 carboxylase/oxygenase is a complex event resulting from the contributions of, at least, three different processes: (a) protein unfolding favored by surface adsorption; (b) oxidation of some cysteine residues; and (c) proteolysis by contaminant proteinases (Rosichan and Huffaker, 1984). This latter contribution has been shown to be minor in the case of purified RuP2 carboxylase/oxygenase of Citrus (Pefiarrubia and Moreno, 1987). Proteins are potentially able to protect the RuP2 carboxylase/oxygenase at all three levels competing for adsorption sites, buffering the oxidative activity and acting as alternative substrate for proteinases. This might explain the extraordinary efficiency in preventing the carboxylase decay achieved by some of them such as hemoglobin, serum albumin, and aldolase, which are clearly effective protective agents for

Pefiarrubia and Moreno

RuP2 carboxylase/oxygenase. Other exogenous proteins may lack some or all of these properties, or display them to a higher or lesser extent showing different degrees of partial protection. In addition, proteins that bind to surfaces will produce a concomitant increase of the initial carboxylase activity. Other procedures for preventing activity decay during storage have been recently proposed (Heuer and Portis, 1990), but they involve low temperatures (either 4°C or freezing with liquid N2) after pretreatment with Mg 2+ and bicarbonate. The use of protective proteins may meet the requirement of preserving activity in experiments where RuP2 carboxylase/oxygenase is incubated at temperatures that are physiologically meaningful. ACKNOWLEDGMENTS

The authors wish to acknowledge the support provided by a grant (PB87-0353) of the CAYCIT and by a fellowship of the Spanish Ministero de Educaci6n y Ciencia (awarded to L.P.). REFERENCES Ackers, G. (1975). In The Proteins, Neurath, H., and Hill, R. L., eds., Vol. I, Academic Press, New York, pp. 293411. Ashani, Y., and Catravas, G. N. (1980). Anal. Bioehem. 109, 55-62. Conejero, V., and Semancik, J. S. (1977). Phytopathology 67, 1424-1426. Creighton, T. E. (1980). Nature 284, 487489. Edmondson, D. L., Badger, M. R., and Andrews, T. J. (1990). Plant Physiol. 93, 1390-1397. Gezelius, K., and Widell, A. (1986). Physiol. Plant. 67, 199-204. Hall, N. P., McCurry, S. D., and Tolbert, N. E. (1981). Plant Physiol. 67, 1220-1223. Heuer, B., and Portis Jr., A. R. (1990). Plant Phisiol. 93, 1511 1513. Jocelyn, P. C. (1972). Biochemistry of the SH Group, Academic Press, New York. Macritchie, F. (1978). Adv. Protein Chem. 32, 283-326. Makino, A., Mae, T., and Ohira, K. (1983). Plant Cell Physiol. 24, 1169-1173. McCurry, S. D., Gee, R., and Tolbert, N. E. (1982). Methods Enzymol. 90, 515-528. Miziorko, H. M., and Lorimer, G. H. (1983). Annu. Rev. Bioehem. 57, 507 535. Pefiarrubia, L., and Moreno, J. (1987). Biochem. Biophys. Acta 916, 227-235. Pefiarrubia, L., and Moreno, J. (1988). Phytochemistry 27, 1999 2004. Pefiarrubia, L., and Moreno, J. (1990). Arch. Biochem. Biophys. 281,319-323. Rosichan, J. L., and Huffaker, R. C. (1984). Plant Physiol. 75, 74~77. Servaites, J. C, (1985). Arch. Biochim. Biophys. 238, 154-160. Suelter, C. H., and DeLuca, M. (1983). Anal. Biochem. 135, 112-119. Tenaud, M., and Jacquot, J. P. (1987). J. Plant Physiol. 16, 1049-1060.

Protection and enhancement of ribulose 1,5 bisphosphate carboxylase activity by exogenous proteins.

When assayed in vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase oxygenase is both enhanced and protected from s...
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