Number Of SH Groups Research Articles

ИЗУЧЕНИЕ ПРОТЕКТОРНОГО ДЕЙСТВИЯ КОРМОВОЙ ДОБАВКИ «ФИТОДОК НЕЙРО» ПРИ СОВМЕСТНОМ ПОСТУПЛЕНИИ СВИНЦА И КАДМИЯ В ОРГАНИЗМ БЕЛЫХ КРЫС

Feed additives containing biologically active substances are used more to neutralize the negative effects of various toxicants, stress, age-related changes in the brain, and also to increase the cognitive abilities of animals. Substances involved in metabolism and activating defense mechanisms are of great interest. As a means to reduce the negative effects of chronic intake of cadmium and lead, the functional feed additive «Phytodoc neuro» was taken, which contains glycine, vitamin E, lecithin and carnitine base as active ingredients. The effectiveness of the feed additive «Phytodoc neuro» at a dose of 4 g/kg of feed as a means to reduce cadmium and lead intoxication when they were simultaneously supplied in doses of 5 and 50 mg/kg of feed, respectively, in terms of metal was studied. With alimentary intake of cadmium and lead in animals, the content of hemoglobin and erythrocytes in peripheral blood significantly decreased, the neutralizing function of the liver was disrupted, there was a clear trend towards a decrease in horizontal and vertical working capacity, body weight, immunoglobulin levels and the number of SH groups in blood serum. The use of the FA «Phytodoc neuro» led to the normalization of all the changed indicators.

Heat-induced interaction between egg white protein and wheat gluten

Some wheat-based food systems, such as cakes and cookies, include mixtures of gluten and egg white protein (EWP) and are processed under heating conditions. Changes in these proteins during processing can affect the quality of the end product. This study investigated protein polymerization during heating of (mixtures of) wheat gluten and EWP. Chemical changes were studied by size-exclusion high performance liquid chromatography (SE-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), thiol (SH) measurement and Fourier transform infrared (FTIR). During heating, protein polymerization was observed in the mixtures of gluten, glutenin, gliadin and EWP according to SE-HPLC profiles and results of SDS-extractable protein. The results of SDS–PAGE profiles of different proteins were in accordance with SE-HPLC. The number of SH groups in the majority of proteins showed a significant decrease, implying that disulfide (SS) bonds contributed to the extractability loss. In addition, changes of secondary structure tested by FTIR indicated protein aggregation.

Aronia melanocarpa treatment and antioxidant status in selected tissues in Wistar rats.

Aronia juice is considered to be a source of compounds with high antioxidative potential. We conducted a study on the impact of compounds in the Aronia juice on oxidative stress in plasma and brain tissues. The influence of Aronia juice on oxidative stress parameters was tested with the use of a model with a high content of fructose and nonsaturated fats. Therefore, the activity of enzymatic (catalase, CAT, and paraoxonase, PON) and nonenzymatic (thiol groups, SH, and protein carbonyl groups, PCG) oxidative stress markers, which indicate changes in the carbohydrate and protein profiles, was marked in brain tissue homogenates. Adding Aronia caused statistically significant increase in the CAT activity in plasma in all tested diets, while the PON activity showed a statistically significant increase only in case of high fat diet. In animals fed with Aronia juice supplemented with carbohydrates or fat, statistically significant increase in the PON activity and the decrease in the CAT activity in brain tissue were observed. In case of the high fat diet, an increase in the number of SH groups and a decrease in the number of PCG groups in brain tissue were observed.

Oriental Plant Alkanna Orientalis Extracts Effects on Enterococcal Membrane-Associated Properties

The oriental plant Alkanna orientalis tissues extracts have been shown to have antimicrobial activity against a number of gram-positive bacteria, particularly Enterococcus hirae, however mechanisms of this activity is not clear enough.A. orientalis intact root and callus cells extracts influence on E. hirae ATCC9790 whole cells H+/K+ exchange, membrane vesicles accessible SH-groups number and ATPase activity was investigated. It was shown that 50 and 100 μl root extract inhibited H+ release ∼1.27 and ∼1.47 fold and K+ accumulation ∼1.25 and ∼1.36 fold, respectively. Whereas, callus cells extract had a stronger effect on ions fluxes rates. The inhibitor of proton translocating F0F1-ATPase, N,N'-dicyclohexylcarbodiimide (DCCD) - sensitive H+ efflux were observed upon addition of both extracts. The number of SH-groups was lowered ∼1.7 and 2 fold compared with the control in the presence of root and callus cells extracts, respectively. The strongest effect was in the case when these substances were added in the assay medium together with 0.2 mM DCCD. The ATPase activity was also decreased ∼3.1 and 3.5 fold compared with the control in the presence of 100 μl intact root and callus cells extracts, respectively. Maximum inhibition of ATPase activity was detected again when intact root and callus cells extracts were added in the assay medium with 0.2 mM DCCD simultaneously.The results point out that A. orientalis intact root and callus cells extracts can directly affect enterococcal membrane-associated FoF1-ATPase, which can be considered as a target for antimicrobial activity.

The Effects of Copper (II) Ions on Enterococcus hirae Cell Growth and the Proton-Translocating FoF1 ATPase Activity

Enterococcus hirae grow well under anaerobic conditions at alkaline pH (pH 8.0) producing acids by glucose fermentation. Bacterial growth was shown to be accompanied by decrease of redox potential from positive values (approximately +35 mV) to negative ones (approximately -220 mV). An oxidizer copper (II) ions (Cu(2+)) affected bacterial growth in a concentration-dependent manner (within the range of 0.05 mM to 1 mM) increasing lag phase duration and decreasing specific growth rate. These effects were observed with the wild-type strain ATCC9790 and the atpD mutant strain MS116 (with absent beta subunit of F(1) of the F(o)F(1) ATPase) both. Also ATPase activity and proton-potassium ions exchange were assessed with and without N,N'-dicyclohexylcarbodiimide (DCCD), inhibitor of the F(o)F(1) ATPase. In both cases (DCCD +/-), even low Cu(2+) concentrations had noticeable effect on ATPase activity, but with less visible concentration-dependent manner. Changes in the number of accessible SH-groups were observed with E. hirae ATCC9790 and MS116 membrane vesicles. In both strains Cu(2+) markedly decreased the number of SH-groups in the presence of K(+) ions. The addition of ATP increased the amount of accessible SH-groups in ATCC9790 and decreased this number in MS116; Cu(2+) blocked ATP-installed increase in SH-groups number in ATCC9790. H(+)-K(+)-exchange of bacteria was markedly inhibited by Cu(2+), but stronger effects were detected together with DCCD. Moreover, discrimination between Cu(2+) and other bivalent cation--Ni(2+) was shown. It is suggested that Cu(2+) ions inhibit E. hirae cell growth by direct affect on the F(o)F(1) ATPase leading to conformational changes in this protein complex and decrease in its activity.

Copper (II) Ions Affect Escherichia coli Membrane Vesicles’ SH-Groups and a Disulfide-Dithiol Interchange Between Membrane Proteins

The SH-groups in Escherichia coli membrane vesicles, prepared from cells grown in fermentation conditions on glucose at slightly alkaline pH, have a role in the F0F1-ATPase operation. The changes in the number of these groups by ATP are observed under certain conditions. In this study, copper ions (Cu2+) in concentration of 0.1 mM were shown to increase the number of SH-groups in 1.5- to 1.6-fold independent from K+ ions, and the suppression of the increased level of SH-groups by ATP was determined for Cu2+ in the presence of K+. Moreover, the increase in the number of SH-groups by Cu2+ was absent as well as the inhibition in ATP-dependent increasing SH-groups number by Cu2+ lacked when vesicles were treated with N-ethylmaleimide (NEM), specific thiol-reagent. Such an effect was not observed with zinc (Zn2+), cobalt (Co2+), or Cu+ ions. The increased level of SH-groups was observed in the hycE or hyfR mutants with defects in hydrogenases 3 or 4, whereas the ATP-dependent increase in the number of these groups was determined in hycE not in hyfR mutants. Both changes in SH-groups number disappeared in the atp or hyc mutants deleted for the F0F1-ATPase or hydrogenase 3 (no activity of hydrogenase 4 was detected in the hyc mutant used). A direct effect of Cu2+ but not Cu+ on the F0F1-ATPase is suggested to lead to conformational changes or damaging consequences, increasing accessible SH-groups number and disturbing disulfide-dithiol interchange within a protein-protein complex, where this ATPase works with K+ uptake system or hydrogenase 4 (Hyd-4); breaks in disulfides are not ruled out.

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 microM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein-diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 microM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 microM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).

Characterization of freeze–dried egg melange long stored after irradiation

During the 4-year period of storage at room temperature of the freeze–dried (control group) and the freeze–dried and gamma-irradiated (2.0 and 3.5 kGy) whole hen's egg mélange, no significant changes were found into the sensory and functional characteristics till the 28th month. The change in the number of SH groups was not unidirectional up to the 28th month and then it started to decrease in all investigated samples. During the entire period of investigation the amount of malondialdehyde in all three groups of egg mélange was considerably below the allowed limit for foodstuffs. The most significant fractions of the protein spectra showed a general tendency of decrease during the storage.

Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD(+) +NADH) to the vesicles caused a approximately 4-fold or approximately 1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD(+) +NADH both were added. The change was lacking in the presence of the F0F1-ATPase inhibitors N,N'-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F0F1-ATPase and, in addition, it was less in potassium ion-free medium. These results are correlated with data about K+-dependent F0F1-ATPase activity, suggesting a relationship between the F0F1-ATPase and K+ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.

Spectral and photosensitizing properties of bacteriochlorin a and its monomethyl and dimethyl esters

We have carried out a comparative study of the photosensitizing activity of bacteriochlorin a (BCA) and its methylated derivatives: monomethyl and dimethyl esters of BCA (BCA MME and BCA DME). We have shown that BCA and its derivatives are bound to the erythrocyte membranes in monomolecular form, and upon photoexcitation in the region of their long-wavelength Qy absorption band, they sensitize oxidation of the major membrane components: proteins and lipids. We have established that despite greater binding of the methylated BCA derivatives to the membranes, their specific activity in photosensitization of peroxide oxidation of membrane lipids and membrane protein tryptophanyls is lower than for BCA. At the same time, BCA MME and BCA DME sensitize oxidation of a larger number of SH groups in membrane proteins, which suggests methylated BCA is predominantly localized in the erythrocyte membrane close to the protein sulfhydryl groups. We also show that the rate of erythrocyte photohemolysis sensitized by BCA MME and BCA DME is significantly faster than for sensitization by BCA. This correlates with the lipophilicity of BCA, BCA MME, and BCA DME, which is responsible for the ability of bacteriochlorins to bind to cell membranes and cells.

Sulfhydryl-Selective, Covalent Labeling of Biomolecules with Transition Metallocarbonyl Complexes. Synthesis of (η5-C5H5)M(CO)3(η1-N-Maleimidato) (M = Mo, W), X-ray Structure, and Reactivity Studies

The photochemical reaction of (eta5-C5H5)Mo(CO)3I with maleimide in the presence of diisopropylamine yielded complex (eta5-C5H5)Mo(CO)3(eta1-N-maleimidato) 4 in 52% yield. The single-crystal X-ray structure of this complex was determined and shows unusual interactions between oxygen atoms of the maleimidato ligand and carbon atoms of the cis-CO ligands. The tungsten analogue of 4, (eta5-C5H5)W(CO)3(eta1-N-maleimidato) 5, was synthesized in 37% yield by the reaction of (eta5-C5H5)W(CO)3I with the thallium(I) salt of maleimide. Complexes 4 and 5 reacted with cysteine ethyl ester and glutathione to afford products of the addition of the sulfhydryl group to the ethylenic bond of the maleimidato ligand. The reaction of 4 and 5 with glutathione proceeded faster than the reaction of the analogous complex (eta5-C5H5)Fe(CO)2(eta1-N-maleimidato) (3). However, all these complexes react with glutathione more slowly than N-ethylmaleimide. Complexes 4 and 5 were used for labeling of bovine serum albumin (BSA), enriched in thiol groups by reaction with Traut's reagent. Reaction of thiolated BSA containing 7.4 SH groups with 4 and 5 gave bioconjugates bearing 6.9 and 6.4 metallocarbonyl moieties, respectively. Under the same conditions, reaction with 3 afforded a BSA conjugate containing 7.6 metallocarbonyl moieties. Labeling was presumed to be site-specific, as the number of metallocarbonyl entities matched very well with the initial number of SH groups measured for the thiolated BSA sample. IR spectra of BSA labeled with 4 and 5 show intense nu(C[triple bond]O)) bands (2042 and 1948 cm(-1) in the latter case), enabling sensitive detection of the bioconjugates in biological samples. Complexes 4 and 5 (especially the latter) should be of interest as heavy atom phasing reagents for protein X-ray crystallography.

Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection: Effect of the denaturation on the determination of thiolic proteins

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC–CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0moldm−3 urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg2+, present in solution as Hg2+–urea complex, is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC–CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10−10–10−12moldm−3, depending on the considered protein, with linear calibration curves spanning over four decades of concentration.

Radioisotopic study of CoMo/Al 2O 3 sulfide catalysts for HDS : Part I. Active site monitoring

In the course of systematic radioisotopic tests of sulfided CoMo catalysts in the HDS of thiophene the effects of catalyst composition and the nature of the feed on the number, distribution and functioning of the active sites of these catalysts have been studied. A method to estimate the ratio between surface SH groups, coordinatively unsaturated sites (CUSs) and functioning vacancies in the catalyst active phase has been established. It has been shown that the CoMo catalyst activity is determined not only by the number of SH groups and their mobility, but also by the number of the vacancies for thiophene adsorption. On the basis of the obtained data, general conditions for the formation and functioning of active sites due to the nature of crude have been formulated and criteria to evaluate the efficiency of catalyst functioning have been developed. The results of pilot plant HDS tests of ten commercial CoMo catalysts have confirmed the developed criteria.

Introduction to serial reviews on oxidative DNA damage and repair

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 μM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1 h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24 h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein–diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 μM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 μM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).

Interaction of perindoprilat with human red blood cells

The effects of perindoprilat on the morphology and dynamic properties of human erythrocytes were studied by light microscopy, electron spin resonance spectroscopy and spectrophotometric methods. Erythrocytes were exposed to perindoprilat at 37 degrees C for 30 and 120 min. It was shown that the drug at a concentration of 0.75 microg/ml did not cause significant changes in the structure of erythrocyte membranes. Higher doses of the drug (7.5 and 75 microg/ml) induced changes in membrane fluidity in the hydrophobic core of the lipid bilayer, the conformation of membrane proteins, the number of SH groups and the activity of membrane-bound acetylcholinesterase (AChE). These modifications were accompanied by changes in the shape of erythrocytes and did not depend on time of incubation. Therefore, it is proposed that perindoprilat perturbs the lipid bilayer and disturbs the organization of the protein-lipid environment.

The mitochondrial oxoglutarate carrier protein contains a disulfide bridge between intramembranous cysteines 221 and 224

The oxoglutarate carrier (OGC) purified from bovine heart mitochondria was treated, both in its active and in its SDS-denatured state, with the fluorescent N-(1-pyrenyl)maleimide and other SH reagents before and after reduction with dithioerythritol or β-mercaptoethanol. The number of SH groups per OGC polypeptide chain was found to be about 1 for the oxidized carrier and 3 for the reduced carrier. The bovine oxoglutarate carrier contains three cysteines: Cys-184, Cys-221 and Cys-224. Sequencing of BrCN cleavage products of oxoglutarate carrier showed that N-(1-pyrenyl)maleimide binds to only Cys-184 of the oxidized protein and also to Cys-221 and Cys-224 after reduction of the protein. These results show the presence of a disulfide bridge between the latter two cysteines of the purified carrier. The oxidized and the reduced forms of the oxoglutarate carrier exhibited different V max but virtually the same K m values for oxoglutarate.

Activity of ribulose bisphosphate carboxylase/oxygenase from potato cultivars with differential response to ozone stress.

The effect of ozone (O3 ) on the carboxylation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the fifth youngest leaf of four cultivars of Solarium tuberosum L. (potato) was investigated. Ozone-tolerant cultivars 'Superior' (SP) and 'Norgold Russet' (NR), and O3 -sensitive cultivars 'Cherokee' (CK) and 'Norland' (NL) were exposed to O3 for five consecutive days in controlled-environment chambers. Leaf samples were obtained during the O3 -exposure period and at 1, 5, and 10 d post-exposure. For both charcoal-filtered air (control) and O3 -treated foliage, total activity of Rubisco declined with increasing leaf age. Ozone induced an accelerated decline in the carboxylation activity of Rubisco in leaves obtained from CK and NL by the third day of O3 exposure. In contrast, O3 had no impact on the Rubisco activity from leaves of SP and NR as compared to non-treated foliage. Differential foliar conductance could not account for the response of the sensitive and tolerant cultivars. Rubisco was purified from cultivars SP and CK to determine whether protein sulphydryl content correlated with foliar O3 sensitivity. The number of SH groups did not differ between the two cultivars, either in chemically denatured or non-denatured Rubisco. Consequently, neither number nor availability of SH groups appear to be factors controlling sensitivity of Rubisco to oxidative damage by O3 .

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809–7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809–7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508–3512]. Identification of the sequence encoding a human WRS is based on ( i) the presence of ‘HIGH’ and ‘KMSKS’ structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203–206]; ( ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089–1091] and deduced from the cDNA seqeuence (six in both cases); ( iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089–1091] and the predicted structure in two different regions.

Effect of osmotic stress on protein turnover in Lemna minor fronds

Evidence is presented that although many proteins from the fronds of Lemna minor L. undergo enhanced degradation during osmotic stress, ribulose-1,5-bisphosphate carboxylase (RuBPCase) is not degraded. Instead RuBPCase is converted in a series of steps to a very high-molecular-weight form. The first step involves the induction of an oxidase system which after 24 h of stress converts RuBPCase to an acidic and catalytically inactive form. Subsequently, the oxidised RuBPCase protein is gradually polymerized to a number of very large aggregates (molecular weight of several million).The conversion of RuBPCase to a high-molecular-weight form appears to be correlated with (i) a reduction in the number of-SH residues and (ii) the susceptibility to in-vitro proteolysis. Indeed, the number of-SH groups per RuBPCase molecule decreases from 89 in the native enzyme to 54 and 22 in the oxidised and polymerized forms, respectively. On the other hand, the oxidised enzyme is more susceptible to in-vitro proteolysis than the native form. However, it is the polymerized form of RuBPCase which is particularly susceptible to in-vitro proteolysis.Western-blotting experiments and anti-ubiquitin antibodies were used to detect the presence of ubiquitin conjugates in extracts from osmotically stressed Lemna fronds. The possible involvement of ubiquitin in the formation of the aggregates is discussed.