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PubMed1991

The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase.

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Alkylation↗

PubMed1990

The interaction of ethanol and zinc on hepatic glutathione and glutathione transferase activity in mice.

The effects of ethanol and/or zinc sulphate on liver glutathione and glutathione transferase activity were studied in mice. Ethanol suppressed glutathione transferase activity and had no significant effect on glutathione levels in the organ. Zinc sulphate administration dose-dependently increased glutathione transferase activity but did not affect hepatic glutathione content. Furthermore, the depressive action of ethanol on glutathione transferase activity was prevented by zinc sulphate pretreatment. It is suggested that zinc sulphate can reactivate glutathione transferase which in turn increases the excretion of the active metabolites produced by ethanol, through conjugation with glutathione in the liver. This action of zinc may alleviate the hepatic toxicity of ethanol in mice.

Animals↗

PubMed1987

alpha, beta-Dihydroxyisovalerate dehydratase. A superoxide-sensitive enzyme.

Increasing the intracellular flux of O-2 by incubating aerobic Escherichia coli with paraquat or plumbagin markedly lowered the alpha, beta-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells. This effect was not seen in the absence of dioxygen and was exacerbated by inhibiting protein biosynthesis with chloramphenicol. These effects of paraquat and of plumbagin were both time- and concentration-dependent. Transfer of E. coli from aerobic to anaerobic conditions caused a rebound of the dehydratase activity, in the continued presence of paraquat and of chloramphenicol, indicating the presence of a mechanism for reactivating this enzyme. The instability of the dehydratase activity in cell extracts was exacerbated by selective removal of superoxide dismutase, but not of catalase, by immunoprecipitation. Addition of exogenous superoxide dismutase reversed the effect of immunoprecipitation; whereas catalase or inactive superoxide dismutase were ineffective. We conclude that the dehydratase is inactivated by O-2. This could account for the bacteriostatic effects of dioxygen and of paraquat.

Catalase↗

PubMed1999

First molecular characterization of a uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1.

Uridine diphosphate galacturonate 4-epimerases (UDPGLEs) are enzymes that convert UDP-glucuronate into UDP-galacturonate. Although the presence of UDPGLEs has been reported in prokaryoic and eukaryotic organisms, the genes coding for these enzymes are completely unknown. The galacturonic acid-containing capsular polysaccharide of Streptococcus pneumoniae type 1 is synthesized through the action of a specific UDPGLE. We have constructed a defined deletion mutant in the cap1J gene (one of the 15 cap1 genes responsible for the synthesis of the type 1 capsule) that exhibited an unencapsulated phenotype. This mutant was unable to synthesize UDPGLE, suggesting that Cap1J was the type 1-specific UDPGLE of S. pneumoniae. Escherichia coli cells harbouring the recombinant plasmid pRMM38 (cap1J) overproduced a 40 kDa protein, characterized as Cap1J on the basis of the N-terminal amino acid sequence analysis, and expressed high levels of enzymatically active Cap1J epimerase. Cap1J was partially purified, although purification to electrophoretic homogeneity inactivated the enzyme irreversibly. The enzyme has the following characteristics: K(m) for UDP-glucuronate, 0.24 mM; pH optimum, 7.5; equilibrium constant (in the direction of UDP-galacturonate formation), 1.3; and an approximate M(r) of 80,000 for the active form. The Cap1J protein exhibited a fluorescence emission spectrum similar to that of NADH. Upon inactivation with p-hydroxymercuribenzoate, the addition of NAD+ and 2-mercaptoethanol were sufficient to reactivate the enzyme. Among several compounds tested, UDP-galactose and UDP-xylose exhibited the highest inhibition of the UDPGLE activity. Inactivation of UDPGLE activity was also observed in the presence of UMP and several reducing sugars. To our knowledge, this is the first example of a thoroughly molecular characterization of a UDPGLE.

Amino Acid Sequence↗

PubMed2001

Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes.

We investigated the role of nitric oxide (NO) in the control of vascular endothelial growth factor A (VEGF) gene expression in cultured human articular chondrocytes. Cell treatment with the NO-generating compound nitrosoglutathione (GSNO) caused a significant accumulation of 4.4 kb VEGF mRNA, a major VEGF mRNA isoform expressing in chondrocytes. This is the first demonstration that NO can induce VEGF mRNA expression in chondrocytes. VEGF mRNA level was not affected in cells exposed to dibutyryl cGMP, a non-hydrolyzable analog of cGMP, suggesting that the cGMP system is not involved in NO-dependent transcriptional activation of VEGF gene. The GSNO-stimulated induction of VEGF mRNA was slightly attenuated by MAP protein kinase inhibitors PD98058 and SB203580, but was completely blocked in cells incubated with GSNO in the presence of catalase and superoxide dismutase, enzymes scavenging reactive oxygen species (ROS), or in the presence of thiol-containing antioxidants, N-acetyl cysteine and reduced glutathione. These results suggest that in articular chondrocytes the GSNO-induced VEGF gene transcriptional activation is dependent on endogenous ROS production and oxidative thiol modifications.

Antioxidants↗

PubMed1997

Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light.

The reaction of two iron-containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp. R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases. Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO-synthase. Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity. Both iron-containing NHases have a high affinity for NO, similar to that of methemoglobin.

Animals↗

PubMedDate not supplied

Oxime-induced reactivation of carboxylesterase inhibited by organophosphorus compounds.

A structure-activity analysis of the ability of oximes to reactivate rat plasma carboxylesterase (CaE) that was inhibited by organophosphorus (OP) compounds revealed that uncharged oximes, such as 2,3-butanedione monoxime (diacetylmonoxime) or monoisonitrosoacetone, were better reactivators than cationic oximes. Cationic oximes that are excellent reactivators of OP-inhibited acetylcholinesterase, such as pyridine-2-aldoxime or the bis-pyridine aldoximes, HI-6 and TMB-4, produced poor reactivation of OP-inhibited CaE. The best uncharged reactivator was 2,3-butanedione monoxime, which produced complete reactivation at 0.3 mM in 2 h of CaE that was inhibited by phosphinates, alkoxy-containing phosphates, and alkoxy-containing phosphonates. Complete reactivation of CaE could be achieved even after inhibition by phosphonates with highly branched alkoxy groups, such as sarin and soman, that undergo rapid aging with acetylcholinesterase. CaE that was inhibited by phosphonates or phosphates that contained aryloxy groups were reactivated to a lesser extent. The cause of this decreased reactivation appears to be an oxime-induced aging reaction that competes with the reactivation reaction. This oxime-induced aging reaction is accelerated by electron-withdrawing substituents on the aryloxy groups of phosphonates and by the presence of multiple aryloxy groups on phosphates. Thus, reactivation and aging of OP-inhibited CaE differ from the same processes for OP-inhibited acetylcholinesterase in both their oxime specificity and inhibitor specificity and, presumably, in their underlying mechanisms.

Animals↗

PubMed2004

Molecular dynamics studies of ground state and intermediate of the hyperthermophilic indole-3-glycerol phosphate synthase.

Indole-3-glycerol phosphate synthase catalyzes the terminal ring closure step in tryptophan biosynthesis. In this paper, we compare the results from molecular dynamics (MD) simulations of enzyme-bound substrate at 298, 333, 363, and 385 K and the enzyme-bound intermediate at 385 K, solvated in TIP3P water box with a CHARMM force field. Results from MD simulations agree with experimental studies supporting the observation that Lys-110 is the general acid. Based on its location in the active site during the MD simulations, Glu-210 warrants classification as the general base instead of the previously proposed Glu-159. We find that the relative population of the reactive enzyme-substrate Michaelis conformers [near attack conformers (NACs)] with temperature correlates well (correlation coefficient of 0.96) with the relative activity of this thermophilic enzyme. At higher temperature, the enzyme-substrate electrostatic interaction favors the binding of the substrate in NAC conformation, whereas, at lower temperature, the substrate is distorted and bound in a nonreactive conformation. This change is reflected in the approximately 1,100-fold increase in population of NACs at 385 K relative to 298 K. The easily determined population of NACs at given temperature tells much about the thermophilic property of the enzyme. Thus, the hyperthermophilic enzyme has evolved to have optimum activity at high temperatures, and, with lowering of the temperature, the electrostatic interaction at the active site is enhanced and the structure is deformed. This model can be regarded as a general explanation for the activity of hyperthermophilic enzymes.

Indole-3-Glycerol-Phosphate Synthase↗

PubMed1994

An essential histidine residue required for fatty acylation and acyl transfer by myristoyltransferase from luminescent bacteria.

The lux-specific acyltransferases are serine esterases responsible for preferential diversion of myristic acid from fatty acid biosynthesis to the luminescent system. In contrast to other acyltransferases, an acylated enzyme intermediate can readily be detected making it ideal for the study of the mechanism of acyl transfer. Although the transferase readily cleaves acyl carrier protein and acyl-CoA, an alternate more rapid and convenient assay involving the cleavage of p-nitrophenyl acyl esters was developed and applied in these studies. The cleavage of the oxyesters by the transferase was shown to have a similar dependence on fatty acid chain length and organic solvents as the cleavage of thioesters. Using this assay, it could be demonstrated that the Photobacterium phosphoreum transferase was inactivated at pH 6 with diethyl pyrocarbonate at a rate (73 M-1 s-1, 10 degrees C) even faster than that reported for other enzymes with reactive histidyl residues at their active site. Spectral changes during chemical modification as well as restoration of activity by neutral hydroxylamine showed that the loss of activity was associated with modification of a single histidine residue. Replacement of the four histidine residues, conserved in all lux-specific acyltransferases, by asparagine demonstrated that cleavage of both thioesters and oxyesters by the P. phosphoreum acyltransferase as well as acylation of the enzyme was blocked on mutation of His-244 but not the other three conserved histidines (His-12, -52, and -75). These results suggest that the histidine residue near the carboxyl terminus (His-244) may be part of a catalytic triad essential for cleavage of acyl esters and transfer of the acyl group to the enzyme.

Acylation↗

PubMed1984

Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae.

Thirty-two monoclonal antibodies (MoAb) to Mycobacterium tuberculosis H37Rv, M. bovis BCG and M. leprae were produced. The spleen cells of BALB/c mice immunized with sonicated or intact bacilli were fused with Sp2/0-Ag-14 myeloma cells. Many more antibody producing hybridomas were found when M. tuberculosis, rather than M. leprae, was used as the immunogen. The MoAb were characterized by an enzyme immunoassay and immunofluorescence on 16 mycobacterial species. The sodium dodecylsulphate polyacrylamide gel electrophoresis immunoperoxidase assay was used to determine the molecular weight of the antigens detected by the MoAb. Antigens of high, low and intermediate molecular weight were found. Some of the antigens were proteinaceous, others of a glycolipid nature. The immunofluorescence assay proved to be essential for the selection of MoAb since some MoAb reacted only in this assay and not in the enzyme immunoassay. The most specific clones were found in the fusions with spleen cells of mice immunized with intact rather than sonicated bacteria. One MoAb (F29-29) reacted only with M. tuberculosis H37Rv; one (F41-3) only with M. leprae and another (F29-45) reacted with M. tuberculosis and M. gastrii. Several MoAb only reacted with three mycobacterial species: M. tuberculosis, M. kansasii and M. gastrii. Others showed unique patterns of reactivity by enzyme immuno- and immunofluorescence assay. The potential use of the MoAb for the identification of mycobacteria and mycobacterial antigens is discussed.

Animals↗

PubMed1982

Reactivation of glutamine synthetase from Escherichia coli after auto-inactivation with L-methionine-S-sulfoximine, ATP, and Mn2+.

Escherichia coli glutamine synthetase auto-inactivated with L-methionine-S-sulfoximine and ATP can be completely reactivated at pH 3.5-4.6 in 1 M KCl and 0.4 M (NH4)2SO4. Both unadenylylated and adenylylated magnesium and manganese enzymes can be reactivated. Reactivation of fully inactivated enzyme is first order (t1/2 approximately 2 min at pH 4.1 and 37 degrees C) and coincides with the stoichiometric release of 0.95 +/- 0.05 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2.0 +/- 0.2 eq of Mn2+ from each subunit. The rate of reactivation increases with decreasing pH and is proportional to the 3rd to 4th power of the hydrogen ion activity; the protonation of 3-4 carboxylic acid groups/subunit therefore may be required to disrupt the enzyme complex. Reactivation rate also increases with increasing KCl concentrations and temperature, with an Arrhenius activation energy of approximately 26 Kcal/mol, suggesting that some protein structural perturbation is required to disrupt the complex. Upon neutralization os reactivation solutions, the ligands and metal ions recombine with the enzyme resulting in its complete reinactivation. Thus, inactivation of glutamine synthetase at neutral pH is due to the extremely tight, but reversible, binding of L-methionine-S-sulfoximine phosphate, ADP, and Mn2+ (K'A greater than 10(12) M-1 for ADP) to the enzyme. Under certain conditions, ATP or ADP can partially inhibit inactivation. Excess ADP inhibits reinactivation by L-methionine-S-sulfoximine phosphate, and ATP has a complex effect on the rate and extent of the autoinactivation reaction.

Adenosine Triphosphate↗

PubMed2005

Up-regulation of defense enzymes is responsible for low reactive oxygen species in malignant prostate cancer cells.

Reactive oxygen species (ROS) are involved in a diversity of important phenomena in the process of tumor development. To investigate the alterations of oxidative stress and their related systems in tumor progression, a variety of components in the antioxidative stress defense system were examined in prostate cancer cell lines, PC3 and LNCaP. Cell surface molecules involved in metastasis were expressed highly in PC3 cells compared with LNCaP cells, and strong invasion ability was shown in PC3 cells only. ROS level in LNCaP cells was twice higher than that in PC3 cells, although nitric oxide (NO) level was similar between the two cell lines. The content of GSH increased up to about 2-fold in PC3 compared with LNCaP. Activities of glutathione reductase, thioredoxin reductase, and glutathione S-transferase except catalase are significantly higher in PC3 cells than in LNCaP cells. Furthermore, oxidative stress-inducing agents caused down-regulation of GSH and glutathione S-transferase much more significantly in LNCaP cells than in PC3 cells. These results imply that malignant tumor cells may maintain low ROS content by preserving relatively high anti-oxidative capacity, even in the presence of stressful agents.

Antioxidants↗

PubMed1998

Reactivation of glyceraldehyde-3-phosphate dehydrogenase using conjugates of monoclonal antibodies with polyelectrolyte complexes. An attempt to make an artificial chaperone.

The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme.

Animals↗

PubMed1971

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase A-inactivated enzyme by phospholipids and detergents.

Specific degradation of the phospholipid membrane of guinea-pig liver microsomal fraction with phospholipase A inactivated glucuronyltransferase. The inactivation was reversed by phosphatidylcholine and mixed microsomal phospholipid micelles at concentrations similar to those present in intact microsomal preparations. The other commonly occurring phospholipids did not reactivate phospholipase A-treated enzyme. Since the mixed microsomal phospholipids consisted mainly of phosphatidylcholine, it is concluded that the reactivation by phospholipids is phosphatidylcholine-specific. Reactivation was also achieved by low concentrations of the cationic detergents cetylpyridinium chloride and cetyltrimethylammonium bromide. Higher concentrations of these detergents inactivated the glucuronyltransferase activity of intact and phospholipase A-treated microsomal fractions. Anionic detergents were potent inactivators of the glucuronyltransferase activity of untreated and phospholipase A-treated microsomal fractions, whereas non-ionic detergents had little effect on the activity of either preparation. Measurements of the zeta-potentials of the micellar species used in this study showed that no obvious relationship existed between the zeta-potentials and the ability to reactivate glucuronyltransferase. However, high positive or negative zeta-potentials were correlated with the ability of the amphipathic compound to inactivate glucuronyltransferase.

Animals↗

PubMed2006

Mechanistic studies of choline oxidase with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions in which betaine aldehyde is formed as an intermediate. The chemical mechanism for the oxidation of choline catalyzed by choline oxidase was recently elucidated by using kinetic isotope effects [Fan, F., and Gadda, G. (2005) J. Am. Chem. Soc. 127, 2067-2074]. In this study, the oxidation of betaine aldehyde has been investigated by using spectroscopic and kinetic analyses with betaine aldehyde and its isosteric analogue 3,3-dimethylbutyraldehyde. The pH dependence of the kcat/Km and kcat values with betaine aldehyde showed that a catalytic base with a pKa of approximately 6.7 is required for betaine aldehyde oxidation. Complete reduction of the enzyme-bound flavin was observed in a stopped-flow spectrophotometer upon anaerobic mixing with betaine aldehyde or choline at pH 8, with similar k(red) values > or = 48 s(-1). In contrast, only 10-26% of the enzyme-bound flavin was reduced by 3,3-dimethylbutyraldehyde between pH 6 and 10. Furthermore, this compound acted as a competitive inhibitor versus choline. NMR spectroscopic analyses indicated that betaine aldehyde exists predominantly (99%) as a diol form in aqueous solution. In contrast, the thermodynamic equilibrium for 3,3-dimethylbutyraldehyde favors the aldehyde (> or = 65%) over the hydrated form in the pH range from 6 to 10. The keto species of 3,3-dimethylbutyraldehyde is reactive toward enzymic nucleophiles, as suggested by the kinetic data with NAD+-dependent yeast aldehyde dehydrogenase. The data presented suggest that choline oxidase utilizes the hydrated species of the aldehyde as substrate in a mechanism for aldehyde oxidation in which hydride transfer is triggered by an active site base.

Alcohol Oxidoreductases↗

PubMed1995

Superoxide radical and iron modulate aconitase activity in mammalian cells.

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

Aconitate Hydratase↗

PubMed1986

Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum.

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.

Azotobacter↗

PubMed2003

The effect of polyols on the reactivation of guanidium chloride-denatured arginine kinase from shrimp feneropenaeus chinensis muscle.

The influence of glycerol, sorbitol, glucose and sucrose, on the refolding and reactivation courses of guanidine-denatured arginine kinase was studied. Glycerol, sucrose, and sorbitol, but not glucose, could improve the reactivation of the denatured arginine kinase, although in all cases aggregation was inhibited. Size exclusion chromatography showed that misfolded products were still formed during polyol-assisted refolding. The chemical and physical characteristics of polyols might explain the various observations.

Animals↗