The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin.
(1/523)
The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible. (+info)
Preparation and properties of S-cyano derivatives of creatine kinase.
(2/523)
The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5'-dithio-bis(2-nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol-blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8 - 0.9-SH group per subunit. This mono-S-cyano derivative of creatine kinase was found 73% as active as the native unmodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted in the bis-S-cyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme. (+info)
Ca2+, K+-regulated intramolecular crosslinking of S-100 protein via disulfide bond formation.
(3/523)
Reaction of the thiol reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2) with the brain-specific protein S-100 favours stabilization of the quaternary structure of the protein via disulfide bond formation. This process is modulated by those cations (Ca2+ and K+) which are known to affect the conformation of the protein. Ca2+ markedly favours the reaction of S-100 with Nbs2 but inhibits subsequent disulfide bond formation; K+, on the contrary, is much less effective in promoting interaction with Nbs2 but strongly stimulates disulfide bond formation. These findings are interpreted assuming that in presence of Ca2+ the three subunits forming the native S-100 protein have two cysteine residues exposed to the solvent but mismatched to form disulfides while in presence of K+ the sulphydryl groups are in a less accessible position to Nbs2 but suitable for S-S bond formation. Crosslinking of S-100 subunits is characterized by the appearance in dodecylsulphate electrophoresis of two very close protein bands having a molecular weight almost identical to that of the native, undenatured protein but not of higher or lower-molecular weight components. This finding, and the demonstration that both the crosslinked and native S-100 proteins have identical profiles when analyzed by sucrose density centrifugation or gel chromatography indicate that disulfide bond formation occurs among subunits of the same molecule. (+info)
Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component.
(4/523)
Plastidic acetyl-CoA carboxylase (ACCase; EC 6.4.1.2), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase. (+info)
Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity.
(5/523)
The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity. (+info)
Modulation of neuronal and recombinant GABAA receptors by redox reagents.
(6/523)
1. The functional role played by the postulated disulphide bridge in gamma-aminobutyric acid type A (GABAA) receptors and its susceptibility to oxidation and reduction were studied using recombinant (murine receptor subunits expressed in human embryonic kidney cells) and rat neuronal GABAA receptors in conjunction with whole-cell and single channel patch-clamp techniques. 2. The reducing agent dithiothreitol (DTT) reversibly potentiated GABA-activated responses (IGABA) of alpha1beta1 or alpha1beta2 receptors while the oxidizing reagent 5, 5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) caused inhibition. Redox modulation of IGABA was independent of GABA concentration, membrane potential and the receptor agonist and did not affect the GABA EC50 or Hill coefficient. The endogenous antioxidant reduced glutathione (GSH) also potentiated IGABA in alpha1beta2 receptors, while both the oxidized form of DTT and glutathione (GSSG) caused small inhibitory effects. 3. Recombinant receptors composed of alpha1beta1gamma2S or alpha1beta2gamma2S were considerably less sensitive to DTT and DTNB. 4. For neuronal GABAA receptors, IGABA was enhanced by flurazepam and relatively unaffected by redox reagents. However, in cultured sympathetic neurones, nicotinic acetylcholine-activated responses were inhibited by DTT whilst in cerebellar granule neurones, NMDA-activated currents were potentiated by DTT and inhibited by DTNB. 5. Single GABA-activated ion channel currents exhibited a conductance of 16 pS for alpha1beta1 constructs. DTT did not affect the conductance or individual open time constants determined from dwell time histograms, but increased the mean open time by affecting the channel open probability without increasing the number of cell surface receptors. 6. A kinetic model of the effects of DTT and DTNB suggested that the receptor existed in equilibrium between oxidized and reduced forms. DTT increased the rate of entry into reduced receptor forms and also into desensitized states. DTNB reversed these kinetic effects. 7. Our results indicate that GABAA receptors formed by alpha and beta subunits are susceptible to regulation by redox agents. Inclusion of the gamma2 subunit in the receptor, or recording from some neuronal GABAA receptors, resulted in reduced sensitivity to DTT and DTNB. Given the suggested existence of alphabeta subunit complexes in some areas of the central nervous system together with the generation and release of endogenous redox compounds, native GABAA receptors may be subject to regulation by redox mechanisms. (+info)
Chemical rescue of the catalytically disabled clostridial glutamate dehydrogenase mutant D165S by fluoride ion.
(7/523)
The catalytically disabled Asp165-->Ser mutant of clostridial glutamate dehydrogenase shows 100000-fold less activity than the wild-type (WT) enzyme in a standard glutamate oxidation assay and 1000-fold less activity in the reductive-amination reaction. The large reduction in the rate has been attributed to removal of the negative charge and the postulated proton-donor capacity of the aspartate carboxyl group. However, fluoride ion (1 M NaF) causes a 1000-fold activation of the mutant enzyme while simultaneously inhibiting WT activity by 20-fold in the forward reaction. For the reverse reaction, F- (1 M) activates the mutant 4-fold and inhibits WT activity to approx. 64%. The net result when 1 M F- is present is a decrease in the WT:mutant activity ratio from 100000 to 5 for the forward reaction. None of the other halides tested, nor NO3(-), CHCOO- or HCOO-, give comparable activation. Re-activation took 15-30 s under assay conditions, suggesting the possibility of conformational change; CD spectroscopy, however, provided no evidence of a substantial change and kinetics of modification using 5,5'-dithiobis(2-nitrobenzoic acid) suggested only subtle structural rearrangement. This phenomenon is discussed in the light of available information about the structure of the mutant enzyme. It is suggested that the F- ion provides a fixed negative charge at the position of the missing aspartate carboxyl group. Therefore, this appears to be an example of 'chemical rescue'. (+info)
Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme.
(8/523)
Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility. (+info)