Rubisco activase: an enzyme with a temperature‐dependent dual function?

Heat treatment of intact spinach leaves was found to induce a unique thylakoid membrane association of an approximately 40 kDa stromal protein. This protein was identified as rubisco activase. Most of the rubisco activase was sequestered to the thylakoid membrane, particularly to the stroma-exposed regions, during the first 10 min of heat treatment at 42 degrees C. At lower temperatures (38-40 degrees C) the association of rubisco activase with the thylakoid membrane occurred more slowly. The temperature-dependent association of rubisco activase with the thylakoid membrane was due to a conformational change in the rubisco activase itself, not to heat-induced alterations in the thylakoid membrane. Association of the 41 kDa isoform of rubisco activase occurred first, followed by the binding of the 45 kDa isoform to the thylakoid membrane. Fractionation of thylakoid membranes revealed a specific association of rubisco activase with thylakoid-bound polysomes. Our results suggest a temperature-dependent dual function for rubisco activase. At optimal temperatures it functions in releasing inhibitory sugar phosphates from the active site of Rubisco. During a sudden and unexpected exposure of plants to heat stress, rubisco activase is likely to manifest a second role as a chaperone in association with thylakoid-bound ribosomes, possibly protecting, as a first aid, the thylakoid associated protein synthesis machinery against heat inactivation.     

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.     

Catalysis of Ribulosebisphosphate Carboxylase/Oxygenase Activation by the Product of a Rubisco Activase cDNA Clone Expressed in Escherichia coli

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase activity was obtained from a partially purified extract of Escherichia coli transformed with a 1.6-kilobase spinach (Spinacia oleracea L.) cDNA clone. This activity was ATP-dependent. Catalysis of rubisco activation by spinach and cloned rubisco activase was accompanied by the same extent of carboxyarabinitol bisphosphate-trapped ¹⁴CO₂ as occurred in spontaneous activation, indicating that rubisco carbamylation is one facet of the rubisco activase reaction. The CO₂ concentration required for one-half maximal rubisco activase activity was about 8 micromolar CO₂. These observations are consistent with the postulated role of rubisco activase in regulating rubisco activity in vivo.     

A fluorometric study with 1-anilinonaphthalene-8-sulfonic acid (ANS) of the interactions of ATP and ADP with rubisco activase

The interactions of ATP and ADP with rubisco activase purified from spinach were investigated by measuring enhanced fluorescence due to ANS-binding to the protein. Evidence of conformational changes was observed from the differences in the interaction of ANS with rubisco activase in the presence of excess ATP and ADP. Fluorescent changes associated with the titration of a rubisco activase-ANS mixture with ATP and ADP indicated that the binding of ADP to rubisco activase was much tighter than that of ATP. The concentration of Mg2+ and pH had significant effects on the affinities of rubisco activase for ATP and ADP, with higher pH and Mg2+ concentration facilitating the binding of ATP to rubisco activase in the presence of ADP. The physiological implications of the binding characteristics of ATP and ADP with rubisco activase on the light-dark regulation of rubisco are discussed.     

Adenosine triphosphate hydrolysis by purified rubisco activase

Activation of ribulose bisphosphate carboxylase/oxygenase (rubisco) in vivo is mediated by a specific protein, rubisco activase. In vitro, activation of rubisco by rubisco activase is dependent on ATP and is inhibited by ADP. Purified rubisco activase hydrolyzed ATP with a specific activity of 1.5 mumol min-1 mg-1 protein, releasing approximately stoichiometric amounts of ADP and Pi. Hydrolysis was highly specific for ATP-Mg and had a broad pH optimum, with maximum activity at pH 8.0-8.5. ATPase activity was inhibited by ADP but not by molybdate, vanadate, azide, nitrate, or fluoride. Addition of rubisco in either the inactive or activated form had no significant effect on ATPase activity. Incubation of rubisco activase in the absence of ATP resulted in loss of both ATPase and rubisco activation activities. Both activities were also heat labile, with 50% loss in activity after 5 min at 38 degrees C and complete inhibition following treatment at 43 degrees C. Both activities showed a sigmoidal response to ATP concentration, with half-maximal activity at 0.053 mM ATP. Rubisco activation activity was dependent on the concentrations of both ATP and ADP. The results suggest that ATPase activity is an intrinsic property of rubisco activase.     

Purification and assay of rubisco activase from leaves

Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes.     

Sucrose regulates growth and activation of rubisco in tobacco leaves<Emphasis Type="Italic">in vitro</Emphasis>

The influence of sucrose onin vitro growth, chlorophyll content, and rubisco/rubisco activase were studied in tobacco leaves. The most pronounced effect onin vitro growth and the chlorophyll content was found at 4% sucrose. The rubisco content increased with increasing concentrations of sucrose, but a point was reached beyond which the increasing concentrations of sucrose caused an inhibition of this enzyme. The rubisco activity showed patterns of change similar to the rubisco content. These data suggest that sucrose may have an affect on the activation and induction of rubisco and that sucrose can be both a positive effector and negative effector depend on its concentration. The degree of intensity of 55 and 15 kD polypeptides, which were identified as the large and small subunit of rubisco, respectively, by SDS-PAGE analysis at 4% sucrose was significantly higher than that of other treatments, indicating that sucrose had an effect on both subunits. We subsequently examined whether the rubisco content and activity of being induced by sucrose is associated with rubisco activase. The rubisco activase content at 4% sucrose was higher than that of the other treatments. A similar change pattern was also observed in the activity of rubisco activase. The intensity of two 52 and 51 kD polypeptide bands at 4% sucrose was higher than that of corresponding bands of other treatments. The stimulatory and inhibitory effects of rubisco by sucrose seemed to be caused by rubisco activase.     

Activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) by rubisco activase : effects of some sugar phosphates

The activation of purified ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) has been studied in the presence of sugar phosphates, and the effect of rubisco activase on this process determined. During an 11-minute time course at pH 7.7 and 11 micromolar CO₂, the activation of rubisco was strongly inhibited by ribulose-1,5-bisphosphate (4 millimolar), fructose-1,6-bisphosphate (1 millimolar) and ribose 5-phosphate (5 millimolar), but this inhibition was overcome by the addition of rubisco activase and activation then proceeded to a greater extent than spontaneous activation of rubisco. Glycerate 3-phosphate (20 millomolar) slowed the initial rate but not the extent of activation and rubisco activase had no effect on this. The activation of rubisco was shown to be affected by phosphoenolpyruvate (3 millimolar) but not by creatine phosphate (3 millimolar) or ATP (3 millimolar), and the creatine-phosphate/creatine phosphokinase system was used to generate the high ATP/ADP quotients required for rubisco activase to function. ATP was shown to be required for the rubisco activase-dependent rubisco activation in the presence of fructose-1,6-bisphosphate (1 millimolar). It is concluded that rubisco activase has a mixed specificity for some sugar phosphate-bound forms of rubisco, but has low or no activity with others. Some possible bases for these differences among sugar phosphates are discussed but remain to be established.     

Influence of benomyl on photosynthetic capacity in soybean leaves

This investigation was performed to study the influence of benomyl on photosynthetic pigments and enzymes in soybean leaves. Chlorophyll and pheophytin levels were reduced by benomyl 45 days after greening. These results indicate that chlorophylla andb, and pheophytin must be controlled by benomyl. SDS-PAGE analysis showed that 50 and 14.5 kD polypeptides represented as the large and small subunits of rubisco. In the both of these subunits, the band intensity of the control was significantly higher than that after benomyl treatment, indicating that these two subunits are affected by benomyl. Benomyl strongly inhibited both the activity and content of rubisco as its concentration was gradually increased. However, it remains unclear whether this reduction of rubisco level was due to a reduced level of rubisco activase. Two major polypeptides of 46 and 42 kD were identified as rubisco activase subunits by SDS-PAGE. The intensity of these two bands was shown to be higher in the control than after benomyl treatment. These results indicate that the rubisco decrease resulting from increased benomyl concentrations was caused by rubisco activase. A significant decrease in both the activity and content of rubisco activase by benomyl was also observed. These results suggest that the decrease in rubisco level caused by benomyl is accompanied by a decrease in both the activity and content of rubisco activase.     

Rubisco Activase Activity in Spinach Leaf Extracts

Rubisco activase is a chloroplast stromal protein that catalyzesthe activation of ribulose-1,5- bisphosphate carboxylase/oxygenase(rubisco) in vivo. Activation must occur before rubisco cancatalyze the photosynthetic assimilation of CO₂. In leaves,photosynthesis and rubisco activation increase with increasinglight intensity. Techniques are described that allow the activityof rubisco activase to be measured in extracts of spinach (Spinaceaoleracea L.) leaf tissue. In this context, rubisco activaseactivity is defined as the ability to promote activation ofthe inactive ribulose-1,5- bisphosphate-bound rubisco in anATP-dependent reaction. Determination of rubisco activase activityin extracts of dark and light treated leaf tissue revealed thatthe activation state of rubisco activase was independent oflight intensity. ¹Present address: Department of Biological Sciences, 213 Carson-TaylorHall, Louisiana Tech University, Ruston, Louisiana 71272, U.S.A.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

The rate of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO₂ fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.     

UV-B radiation affects chlorophyll and activation of rubisco by rubisco activase in<Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L. leaves

We studied the influence of UV-B radiation on chlorophyll and rubisco activation by rubisco activase in the leaves of jackbean (Canavalia ensiformis). Chlorophyll content was decreased, indicating that the synthesis of those molecules may have been degraded or repressed after exposure. Rubisco content was significantly lower in radiated tissue compared with the untreated control; rubisco activity showed a similar pattern of change. Based on these data, we suggest that rubisco activity is associated with the level of rubisco protein, and that UV-B inhibits its activation and induction, as well as that of rubisco activase. Therefore, we propose that the inhibitory effect of rubisco by UV-B may be caused by rubisco activase.     

Purification and species distribution of rubisco activase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.     

Immunoblot analysis of the expression of genes for barley rubisco activase inE. coli

Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barleyRca genes (RcaA1,RcaA2 andRcaB) were expressed inE. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts ofE. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed inE. coli as active forms of 46 kD and 42 kD.     

Influence of cadmium on rubisco activation in<Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L. leaves

We studied the effect of cadmium on chlorophylls and rubisco activation inCanavalia ensiformis L. leaves. Chlorophyll levels were reduced by 5.0 μM Cd. Rubisco activity at 5.0 μM Cd was significantly smaller than that at no treatment. Rubisco content showed patterns of change similar to rubisco activity. These data suggest that rubisco activity was associated with an amount of rubisco protein, and that the activation and induction of rubisco is inhibited by Cd. The degree of intensity of 50 and 14.5 kD polypeptides identified as the large and small subunit of rubisco by SDS-PAGE analysis at 5.0 μM Cd was significantly lower than that at control, indicating Cd had a effect on both subunits. Under the assumption that effects of Cd on rubisco may be related to rubisco activase, in addition to, its activity and content were determined. The rubisco activase activity at 5.0 μM Cd was more decreased than, the control. A similar change pattern was also observed in content of rubisco activase. Remarkable differences in the intensitiy of both the 45 kD and 41 kD band were found between at control and Cd-treatment. These results suggest that the change in the levels of rubisco activase leads to a subsequent alteration of rubisco levels.     

Decrease in carbamylation of rubisco by high CO2 concentration is due to decrease of rubisco activase in kidney bean

Decrease in rubisco activation at high CO₂ concentration was caused by decrease in carbamylation of rubisco (Rohet al., 1996). However, it is unclear whether decrease in carbamylation rate at high CO₂ concentration is due to decrease in activity itself or content of rubisco activase. To clarify this ambiguity, investigation was performed to determine effects of CO₂ concentration on rubisco activase with kidney bean (Phaseolus vulgaris L.) leaves grown at normal CO₂ (350 ppm) and high CO₂ (650 ppm) concentration. The analysis of Western blotting showed that the 50 and 14.5 kl) polypeptides were identified immunochemically as the large and small subunits of rubisco in the preparation, respectively. For the 14.5 kD small subunit, the degree of intensity at high CO₂ concentration was similar to that at normal CO₂ concentration. For the 50 kD large sububit, however, the intensity of a band at high CO, concentration was significantly higher than that at normal CO₂ concentration, indicating that only the large subunit is affected by high CO₂ concentration. The analysis of Western immunoblotting showed two major polypeptides at 46 and 42 kD which were identified as rubisco activase subunits. The intensities of two bands were shown to be higher at normal CO₂ than high CO₂ concentration. These data indicate that decrease of carbamylation resulting from increase of CO₂ concentration was caused by rubisco activase. Finally, by employing ATP hydrolysis assay and ELISA, we also observed a significant decrease in both activity and content of rubisco activase as CO₂ concentration was raised from normal to high CO₂ concentration. These results suggest that decrease in rubisco carbamylation at high CO₂ concentration is caused by activity itself and/or content of rubisco activase.     

Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes

A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase(rubisco), rubisco activase, washed thylakoid membranes, andATP was used to demonstrate a light-dependent stimulation ofrubisco activation. ATP, ribulose bisphosphate, H⁺, and Mg²⁺concentrations are normally light-dependent variables in thechloroplast but were maintained at pre-determined levels. Resultsindicated that rubisco activase and washed thylakoid membranesare sufficient to catalyze light stimulation of rubisco activationwith the reconstituted system, and that rubisco activase isrequired for this light stimulation. The washed thylakoid membranesdid not exhibit rubisco activase activity, nor was rubisco activaseprotein detected immunologically. Light-dependent activationof rubisco in the reconstituted system was similar in whole-chainand PS I electron transport reactions, and saturated at approximately100 µmol photons m^–2 s^–1. ¹ Present address: Department of Biological Sciences, LouisianaTech University, Ruston, LA 71272, U.S.A.     

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl₂, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg²⁺ at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K_act(CO₂) of rubisco activation in the reconstituted system was 4 micromolar CO₂, compared to a K_act(CO₂) of 25 to 30 micromolar CO₂ for the previously reported spontaneous CO₂/Mg²⁺ activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO₂ and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.