Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

Abstract

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu(2+), benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.

C172S Substitution in the Chloroplast-encoded Large Subunit Affects Stability and Stress-induced Turnover of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

“…After adding 2% (w/v) polyvinylpolypynolidone and stirring for 5 min at 4°C, the crude extract was centrifuged at 35,000 ϫ g for 10 min. Rubisco was purified from the supernatant by ammonium sulfate precipitation, sucrose gradient fractionation, and DEAE-cellulose chromatography as previously detailed (22). The final protein preparation contained Ͼ90% Rubisco (ascertained by densitometry of Coomassie Blue-stained gels) and was essentially free of nucleic acids (A 280/260 nm Ͼ 1.7).…”

“…Purified Rubisco was activated in 100 mM Tris-HCl, pH 8.2, 10 mM MgCl 2 , and 10 mM NaHCO 3 by gel filtration through a Sephadex G-25 column (Amersham Pharmacia Biotech PD-10) and further diluted to an absorbance of 0.31 at 280 nm (ϳ0.2 mg/ml). RuBP carboxylase activity was measured as the incorporation of acid-stable 14 C from NaH 14 CO 3 (22). The amount of total protein in the extracts was determined by the method of Lowry et al (36).…”

“…At the indicated times, 20-l aliquots of the samples were mixed with 180 l of 100 mM Tris-HCl, pH 8.2, 10 mM MgCl 2 , and 10 mM NaHCO 3 in preincubated (30°C) plastic vials (Bio-vial, Beckman Instruments). The mixtures were further incubated at 30°C for 15 min before the RuBP carboxylase assay was started by the addition of RuBP and NaH 14 CO 3 (22). Redox Buffers and Susceptibility to Proteolysis-Aliquots (200 l) of purified Rubisco (0.2 mg/ml) were combined with 100 l of cystamine/ cysteamine mixtures of constant monomeric concentration (120 mM in 100 mM Tris-HCl, pH 8.2, 10 mM MgCl 2 , and 10 mM NaHCO 3 ).…”

“…Then, 55 l of 0.25 M Tris-HCl, pH 6.8, 2 M 2-mercaptoethanol, 6.5% SDS, and 25% (w/v) glycerol were added, and the samples were boiled for 4 min. Aliquots (10 -20 l) were subjected to SDS-polyacrylamide gel electrophoresis, immunoblotting with rabbit antiserum directed against Euglena gracilis Rubisco, and blot densitometry as described previously (17,22).…”

“…Proteolysis of Rubisco is indeed a prominent feature in the stress responses of plants and algae, and several studies have shown that Rubisco is oxidatively modified prior to its degradation (15)(16)(17)(18)(19)(20). Moreover, oxidation of Cys residues in vitro has been proposed to switch Rubisco from a protease-resistant to protease-sensitive form (21)(22)(23). The critical residues have not yet been identified, but because oxidative modification of Rubisco is widespread among species (15)(16)(17)(18)(19)(20), it is likely that these residues are conserved.…”