E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.     

Inhibition of maize leaf phosphoenolpyruvate carboxylase by diethyl oxaloacetate

Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The K_i values, based on total diethyl oxaloacetate, decreased with increasing pH, while the K_i values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine     

Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C(4) Plants : An Effect of pH

Photoactivation of phosphoenolpyruvate carboxylase in C₄ plants is detected more efficiently when activity is assayed at suboptimum pH (e.g. 7.2); the magnitude of the light effect is often larger at low phosphoenolpyruvate concentration.

Darkness and low assay pH induce an allosteric behavior (positive cooperativity with phosphoenolpyruvate) which is relieved in light or by higher pH; thus, normal Michaelis-Menten kinetics are exhibited only when the enzyme is extracted during the day and assayed at pH 8.2.

Light activation, pH, and substrate level appear to be components of a regulatory device suppressing the activity in darkness and enhancing it under light.

     

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K_m for phosphoenolpyruvate and reducing V_max. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K_m for phosphoenolpyruvate, but having little effect on V_max. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K_m for phosphoenolpyruvate and an increased V_max, but the night, or insensitive, form shows only an increase in V_max in response to glucose-6-phosphate.

     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

Involvement of thiol groups in the activity of phosphoenolpyruvate carboxylase from maize leaves

Purified maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) was completely inactivated by several thiol-modifying reagents, including, CuCl₂, CdCl₂ and N-ethylmaleimide. The inactivation by CuCl₂ could be reversed by dithiothreitol, suggesting the involvement of vicinal dithiols in the inactivation process.Complete inactivation of phosphoenolpyruvate carboxylase was correlated with the incorporation of two mol (³H)N-ethylmaleimide per 100-kilodalton subunit. The total protection of the enzyme against N-ethylmaleimide inactivation afforded by the substrate, phosphoenolpyruvate, was correlated with the protection of one mol (³H)N-ethylmaleimide reactive residue per mol subunit.The complete inactivation of phosphoenolpyruvate carboxylase by N-ethylmaleimide and the protection afforded by phosphoenolpyruvate against modification suggest the presence of an essential cysteine residue in the catalytic site of the C₄ leaf enzyme.Abbreviations PEP, phosphoenolpyruvate - Mops, 4-morpholinepropanesulphonic acid (Consejo Nacional de Investigaciones Científicas y Técnicas, Fundación M. Lillo y U.N. de Rosario).     

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5′-dithio-bis-(2-nitrobenzoic acid) and ZnCl₂, which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H₂O₂. SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.     

Evidence for Light-stimulated Synthesis of Phosphoenolpyruvate Carboxylase in Leaves of Maize

Illumination (22,000 lumens per meter²) of etiolated maize plants for 80 hours brings about a 5-fold increase in phosphoenolpyruvate carboxylase activity per unit of protein. An increase in carboxylase protein and incorporation of [³⁵S]methionine into the protein occurs simultaneously with the activity increase. In green plants, the level of phosphoenolpyruvate carboxylase protein and enzyme activity is dependent on the intensity of light during growth. These results are consistent with the conclusion that the activity increase results from light-stimulated de novo synthesis of phosphoenolypyruvate carboxylase protein.     

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone     

Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1

The relationship between activation of the latent ATPase activity of isolated chloroplast coupling factor 1 (CF1) and reduction of a disulfide in the gamma subunit has been assessed. The sulfhydryl residues involved in the disulfide bond are distinct from residues normally accessible to maleimide modification during incubation of thylakoids in the dark or the light. Dithiothreitol-induced activation is time dependent, and correlates with reduction of the disulfide. Sulfhydryl residues exposed during activation can be reoxidized to disulfide by incubation with iodosobenzoate , with a concomitant loss of ATPase activity. Activation and deactivation are reversible, but deactivation is prevented by treatment of the reduced enzyme with N-ethylmaleimide. Heat activation does not reduce the disulfide bond unless dithiothreitol is present during activation. Prior heating of CF1, which partially activates the enzyme, renders the disulfide more susceptible to subsequent dithiol reduction. The activity obtained when heat and dithiothreitol are used together is approximately equal to the sum of the partial activations obtained with heat or dithiothreitol alone. Iodosobenzoate has no effect on heat-activated CF1. Enzyme activated by heating in the presence of dithiothreitol can be partially deactivated, consistent with reversal of the activity attributable to the dithiol effect. Fluorescence polarization of anilinonaphthylmaleimide bound to the reduced enzyme indicates that the sulfhydryl residues involved in the disulfide are in a less rigid environment than the other two sulfhydryl residues in the gamma subunit. Polarization of anilinonaphthylmaleimide bound to these sulfhydryls is reduced by heat treatment of CF1. The increased susceptibility of the disulfide to reduction upon heat treatment, and the activation of ATPase activity with or without disulfide bond cleavage are indicative of conformational changes within the gamma subunit that occur during the conversion of CF1 from a latent to an active ATPase. In addition the results are consistent with at least two distinct conformational forms of CF1 that can hydrolyze ATP.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Escherichia coli phosphoenolpyruvate carboxylase: studies on the mechanism of multiple allosteric interactions.

Some kinetic studies of the interactions between Escherichia coli phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating) EC 4.1.1.31) acetyl coenzyme A, fructose 1,6-bisphosphate, and aspartate were performed. Activation of the enzyme by fructose 1,6-bisphosphate is anomalous by comparison with acetyl coenzyme A in that it confers hysteretic properties on the enzyme. In the presence of both activators and aspartate, hysteresis is observed also, but the approach to optimum catalytic activity can be fit to an equation for a second-order reaction with respect to enzyme concentration. Since, however, hysteresis is not a result of any apparent association-dissociation reaction, the apparent fit to a second-order kinetic equation is probably not real but is the result of a multistep activation mechanism. Hysteresis is not eliminated by preincubation of the enzyme with fructose 1,6-bisphosphate, acetyl coenzyme A, or phosphoenolpyruvate singly or in any pair of combinations. Hysteresis is associated, therefore, with the slow conformation change from the inactive species to the active species under the influence of all three of those reactants. The enzyme complex resulting from the binding of each activator, including phosphoenolpyruvate, has an increased affinity for the other activators. A kinetic method for estimating the relative changes in affinity of these complexes for some of the other reactants is presented. At concentrations of the activators below their K_a, synergistic effects are evident, particularly in their ability to relieve aspartate inhibition. Aspartate inhibition is competitive with acetyl coenzyme A both in the absence and in the presence of low concentrations of fructose 1,6-bisphosphate. Increasing the concentrations of fructose 1,6-bisphosphate results in an increase in the apparent K_l for aspartate, suggesting that synergistic activation by fructose 1,6-bisphosphate is a result of the increased affinity of the fructose 1,6-bisphosphate-enzyme complex for acetyl coenzyme A, and a shift in the concentration of enzyme species away from the one(s) to which aspartate can bind most easily. In the presence of fructose 1,6-bisphosphate alone optimal activation can be achieved, but the concentrations required in vitro are high and suggest that fructose 1,6-bisphosphate alone does not function in that capacity physiologically, but primes the enzyme for more effective activation by acetyl coenzyme A and/or phosphoenolpyruvate.     

Cooperative Effects of Light and Temperature on the Activity of Phosphoenolpyruvate Carboxylase from Amaranthus paniculatus L

The phosphoenolpyruvate carboxylase of Amaranthus paniculatus shows in vitro optimum affinity (S_0.5) to phosphoenolpyruvate at a relatively high temperature (about 35°C); even in the presence of activators, it functions efficiently only above 25 to 27°C. At lower temperatures, a steep increase of activity with temperature is observed, due to the high activation energy for the catalyzed reaction. The same behavior in vivo could amplify the photoactivation of the enzyme to a large extent, since the night/day transition is soon followed by a considerable rise in leaf temperature.     

Redox Regulation of Large Conductance Ca2+-activated K+ Channels in Smooth Muscle Cells

The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca²⁺-activated K⁺ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, β-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2′-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP_o relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP_o following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.     

Alfalfa root nodule carbon dioxide fixation : I. Association with nitrogen fixation and incorporation into amino acids

In vivo CO₂ fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.

Forage harvest and nitrate application reduced both in vivo and in vitro CO₂ fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.

The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.

The results demonstrate that CO₂ fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO₂ fixation for attached and detached alfalfa nodules at low CO₂ concentrations (0.13-0.38% CO₂) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO₂ fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa.

     

Bilevel disulfide group reduction in the activation of c(4) leaf nicotinamide adenine dinucleotide phosphate-malate dehydrogenase

The time course of thioredoxin-mediated reductive activation of isolated Zea mays nicotinamide adenine dinucleotide phosphatemalate dehydrogenase is highly sigmoidal in nature. We examined the factors affecting these kinetics, including the thiol-disulfide status of unactivated and activated forms of the enzyme. The maximum steady rate of activation was increased, and the length of the lag in activation decreased, as the concentrations of thioredoxin-m, dithiothreitol, and KCl were increased. The lag in activation (sigmoidicity) was eliminated by preincubating the unactivated enzyme with 100 mm 2-mercaptoethanol; this pretreatment did not activate the enzyme. Unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase was found to contain approximately two SH groups per subunit, increasing to about four SH per subunit after pretreatment with 2-mercaptoethanol and six SH per subunit after activation by incubating the enzyme with dithiothreitol. We suggest that reduction of one particular higher redox potential disulfide group in unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase facilitates the subsequent reduction of the critical S-S group (regulatory S-S) necessary to generate the active form of the enzyme.     

Modification of an essential amino group of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal phosphate and by pyridoxal phosphate-sensitized photooxidation

Phosphoenolpyruvate carboxylase from maize leaves was inactivated by pyridoxal 5'-phosphate in the dark and in the light. A two-step reversible mechanism is proposed for inactivation in the dark, which involves the formation of a noncovalent complex prior to a Schiff base with amino groups of the enzyme. Spectral analysis of pyridoxal 5'-phosphate-modified phosphoenolpyruvate carboxylase showed absorption maxima at 432 and 327 nm, before and after reduction with NaBH4, respectively, suggesting that epsilon-amino groups of lysine residues are the reactive groups in the enzyme. A correlation between spectral data and the maximal inactivation obtained with several concentrations of inhibitor allowed us to establish that the incorporation of 4 mol of pyridoxal 5'-phosphate per mole of holoenzyme accounts for total inactivation. The absence of modifier bound to phosphoenolpyruvate carboxylase when the modification was carried out in the presence of phosphoenolpyruvate and MgCl2 suggests the existence of an essential lysine residue at the catalytic site of the enzyme. Modification of phosphoenolpyruvate carboxylase in the light under an oxygen atmosphere resulted in an irreversible inactivation, which was completely protected by phosphoenolpyruvate and MgCl2. Spectral analysis of the photomodified enzyme showed an absorption peak of 320 nm, suggesting light-mediated addition of a nucleophilic residue (probably an imidazole group) to the pyridoxal 5'-phosphate-lysine azomethine bond.     

Redox modulation of osmotic water permeability in plasma membranes isolated from roots and shoots of pea seedlings

Partitioning in a biphasic polymer system was used to isolate plasmalemma (PM) from roots and shoots of etiolated pea seedlings. The membrane preparations were used to assess the osmotic water permeability (P _os) with the stopped-flow method. The Western-blot technique was employed to determine the membrane content of the PIP-family of aquaporins, and their activity was estimated by measuring the rate of osmotic vesicle shrinking in the presence of inhibitors, HgCl₂ and AgNO₃. Monobromobimane fluorescent dye was used to determine the quantity of sulfhydryl groups in cell membranes and follow the effect of SH-oxidizing (diamide) and SH-reducing (dithiothreitol and tributylphosphine) agents on P _os of the root PM and oligomerization of aquaporins. The shoot PM was shown to combine high P _os with low aquaporin content. In the root PM, P _os was lower and the aquaporin content greatly exceeded that in the shoots. HgCl₂ and AgNO₃ did not decrease the rate of osmotic shrinking in root membrane vesicles, whereas considerably (by 40–50%) inhibited this index in the shoot membranes. Root and shoot PM preparations dramatically differed in their SH-group contents: the former exceeded the latter sixfold. When added to the homogenization medium, diamide and tributylphosphine affected the content of SH-groups and P _os in the root PM. In the roots, diamide decreased the quantity of SH-groups almost twofold and increased P _os fourfold, and the introduction of tributylphosphine produced a twofold increase in the quantity of SH-groups with only slight decrease in the P _os. Immunological analysis of membranes isolated in the presence of diamide showed that the ratio between the monomer and dimer forms of aquaporins in two membrane preparations depended on the presence of dithiothreitol in the denaturing buffer apparently because dithiothreitol exposed and reduced disulfide bonds essential for monomer interactions and inaccessible for interaction with redox modifiers of SH-groups in the membrane. Because of their inaccessibility, these modifiers could not cause the changes of P _os in the root PM produced by oxidation and reduction of SH-groups. This phenomenon is probably related to the change in the status of SH-groups in two cysteine residues at the N-end of the aquaporin loop C oriented outward into the apoplast.