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PubMed2005

An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium.

An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.

Amino Acid Sequence↗

PubMed1984

Interaction of calmodulin with muscle phosphofructokinase. Changes of the aggregation state, conformation and catalytic activity of the enzyme.

Phosphofructokinase from muscle has been shown to be a calmodulin-binding protein [Mayr, G.W. and Heilmeyer, L.M.G., Jr (1983) FEBS Lett. 159, 51-57]. Details of the influence of calmodulin on the aggregation state, the conformation and the catalytic properties of phosphofructokinase have been studied by enzymatic and light-scattering analyses. Calmodulin acts as a Ca2+-dependent hysteretic inhibitor of the highly active enzyme. At least one mole of calmodulin binds to each protomer of the enzyme, induces a shift from the highly active tetrameric towards an inactive dimeric state and slowly changes the conformation of the dimers. Dissociation of calmodulin from conformationally changed dimers by removal of Ca2+ stops the inactivation. Without a significant regain of catalytic activity large polymers are rapidly formed. For a reactivation of the inactivated enzyme, calmodulin has to remain associated and the incubation conditions must be changed in a way to allow for a back isomerization and reassociation of dimers. The isomerization reaction is promoted by Mg . ATP, the reassociation reaction most effectively by fructose bisphosphate. A model for the calmodulin-phosphofructokinase interaction is proposed.

Adenosine Triphosphate↗

PubMed1991

Chloroplast and mitochondrial mechanisms for protection against oxygen toxicity.

As a consequence of their oxygen rich environment, organelles of photosynthetic tissues are exposed to large fluxes of oxyradicals and reactive oxygen species. Superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen are all potential by-products of respiratory and photosynthetic systems. Strong reductants found in mitochondria and chloroplasts along with a steady flux of photosynthetically generated oxygen enhance the potential for oxyradical production. Unless neutralized by scavenger substrates or enzymes, these reactive intermediates pose a lethal threat. The presence of superoxide dismutases, catalases, various peroxidases and scavenger substrates are all means of defences available to protect organelles. A balance between oxyradical production and neutralization should exist. Perturbations in generation or in sequestration caused by environmental or nutritional factors might profoundly alter the steady state level of oxyintermediates.

Chloroplasts↗

PubMed1997

Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning.

It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.

Acetylcholinesterase↗

PubMed1993

Effect of glutathione on aconitase in Escherichia coli.

The effect of glutathione (GSH) on the superoxide-sensitive [4Fe-4S]-containing aconitase of Escherichia coli was explored. A mutant deficient in GSH biosynthesis, designated gshA, grew slower in a defined medium than did the parental strain and this effect was more pronounced when succinate was supplied as the carbon source in place of glucose. This suggested that the citric acid cycle was compromised in the gshA strain. Aconitase activity was approximately 25% lower in GSH-deficient cells growing on either glucose or succinate, and was lower still in strains producing less superoxide dismutase. Addition of GSH to the medium stimulated growth of the gshA strain on succinate. It also elevated the aconitase activity in the presence of chloramphenicol, which was added to block protein synthesis. Dithiothreitol and 2-mercaptoethanol were much less effective in this regard than was GSH. Exposure of cultures to 4.2 atm O2 caused a rapid decline in aconitase activity and this was the case in both GSH-proficient and GSH-deficient E. coli; however, the reactivation which was seen when the hyperoxic exposure was terminated was significantly impaired in the gshA strain. There is a dynamic balance between inactivation of aconitase by superoxide and reactivation by Fe(II) and this balance is altered in GSH-deficient E. coli. GSH may facilitate reactivation of aconitase, and of other [4Fe-4S]-containing dehydratases, by increasing the rate of transfer of Fe(II) to the [3Fe-4S] site.

Aconitate Hydratase↗

PubMed1992

Glutathione reductase from Saccharomyces cerevisiae undergoes redox interconversion in situ and in vivo.

Redox interconversion of glutathione reductase was studied in situ with S. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 microM NADPH or 100 microM NADH caused 50% inactivation in normal cells in 30 min, 0.75 microM NADPH or 50 microM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP+ concentrations. The inactivation of yeast glutathione reductase has also been studied in vivo. The activity decreased to 75% after 2 hours of growth with glucono-delta-lactone as carbon source, while NADPH rose to 144% and NADPH+ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 microM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD+ and NADPH/NADP+ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-delta-lactone is oxidized promotes in vivo inactivation of glutathione reductase.

Cell Membrane Permeability↗

PubMed2005

Maltol/iron-mediated apoptosis in HL60 cells: participation of reactive oxygen species.

Apoptosis of HL60 cells by maltol was analyzed in relation to the maltol/iron-mediated generation of reactive oxygen species. Addition of maltol with FeSO(4) induced an apoptotic cell death as judged by flow cytometry analysis and DNA fragmentation on electrophoresis, but maltol or iron alone did not affect the cells. Treatment of HL60 cells with maltol/iron complex caused an effective inactivation of aconitase the most sensitive enzyme to reactive oxygen species. Maltol/iron-mediated apoptosis and the inactivation of aconitase was prevented by TEMPOL, the scavenger of reactive oxygen species. These findings suggest that maltol/iron complex can generate reactive oxygen species by the redox cycling, resulting in an apoptosis of HL60 cells. Cytotoxicity of maltol can be explained by the prooxidant properties of this compound.

Aconitate Hydratase↗

PubMed1998

Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor.

Stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H2O2 that is required for growth factor-induced protein tyrosine phosphorylation. The effect of H2O2 produced in response to epidermal growth factor (EGF) on the activity of protein-tyrosine phosphatase 1B (PTP1B) was investigated in A431 human epidermoid carcinoma cells. H2O2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, most likely to sulfenic acid. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. Oxidation by H2O2 prevented modification of the catalytic cysteine of PTP1B by iodoacetic acid, suggesting that it should be possible to monitor the oxidation state of PTP1B in cells by measuring the incorporation of radioactivity into the enzyme after lysis of the cells in the presence of radiolabeled iodoacetic acid. The amount of such radioactivity associated with PTP1B immunoprecipitated from A431 cells that had been stimulated with EGF for 10 min was 27% less than that associated with PTP1B from unstimulated cells. The amount of iodoacetic acid-derived radioactivity associated with PTP1B reached a minimum 10 min after stimulation of cells with EGF and returned to base line values by 40 min, suggesting that the oxidation of PTP1B is reversible in cells. These results indicate that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells and that concurrent inhibition of protein-tyrosine phosphatases by H2O2 might also be required.

Amino Acid Sequence↗

PubMed1992

Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.

Cations, Divalent↗

PubMed1988

Reactivation of creatine kinase activity by preincubation with buffered NAC diluent.

We have shown that several commercial control sera containing reversibly inactivated creatine kinase are reactivated by pre-incubation in N-acetyl cysteine assay diluent. The increase in activity can proceed for up to 4 h. This reactivation is not seen in patients' sera even after storage for 14 days at -20 degrees C, 7 days at 4 degrees C, or 2 days at 20 degrees C, during which time no loss of activity was evident. This study brings into question the validity of using consensus values to standardise enzyme assays.

Acetylcysteine↗

PubMed2004

Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese.

Although, several studies have been reported on the effects of oxidants on the structure and function of other molecular chaperones, no reports have been made so far for the chaperonin GroEL. The ability of GroEL to function under oxidative stress was investigated in this report by monitoring the effects of hydrogen peroxide (H(2)O(2)) on the structure and refolding activity of this protein. Using fluorescence spectroscopy and light scattering, we observed that GroEL showed increases in exposed hydrophobic sites and changes in tertiary and quaternary structure. Differential sedimentation, gel electrophoresis, and circular dichroism showed that H(2)O(2) treated GroEL underwent irreversible dissociation into monomers with partial loss of secondary structure. Relative to other proteins, GroEL was found to be highly resistant to oxidative damage. Interestingly, GroEL monomers produced under these conditions can facilitate the reactivation of H(2)O(2)-inactivated rhodanese but not urea-denatured rhodanese. Recovery of approximately 84% active rhodanese was obtained with either native or oxidized GroEL in the absence of GroES or ATP. In comparison, urea-denatured GroEL, BSA and the refolding mixture in the absence of proteins resulted in the recovery of 72, 50, and 49% rhodanese activity, respectively. Previous studies have shown that GroEL monomers can reactivate rhodanese. Here, we show that oxidized monomeric GroEL can reactivate oxidized rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.

Chaperonin 60↗

PubMed1988

Development and characterization of a syngeneic monoclonal antibody to a rat mammary tumor metastasis-associated mucin-like cell-surface antigen (gp580).

A rat hybridoma producing IgM monoclonal antibody (MAb) GP21:56 was generated with specificity for a high-molecular-weight, mucin-like glycoprotein (gp580) present on highly metastatic 13762NF rat mammary adenocarcinoma cells. The hybridoma was made by fusing rat Y3 Ag1.2.3 myeloma cells with spleen cells from a rat immunized i.d. with purified gp580. The gp580 appeared to be of low immunogenicity in syngeneic F344 rats because a total of 27 fusions were required to produce one hybridoma with specificity for this glycoprotein. Immunoblotting of purified gp580 after electrophoresis in 1% agarose and antibody-binding assays using purified gp580 linked to microtiter plates confirmed that MAb GP21:56 bound specifically to gp580. Other MAbs made against breast mucins were negative for gp580 reactivity. Enzyme-linked immunoabsorbent assays (ELISA) and radiolabelled antibody binding assays demonstrated that MAb GP21:56 bound to 13762NF adenocarcinoma cell lines and clones in relation to their spontaneous metastatic potentials; significantly more MAb GP21:56 bound to highly metastatic MTLn3 cells than to low metastatic MTC cells, and MAb GP21:56 showed little reactivity towards the majority of other cell lines tested, whether of rodent or of human origin. Kinetic binding studies indicated that MAb GP21:56 does not have a high affinity for gp580 but, once bound, it shows high avidity for this sialogalactoprotein. Localization studies using frozen tissue sections of 13762NF tumors indicated that MAb GP21:56 reacts with tumor cells grown in vivo in an analogous manner to in vitro cultured cells. Using immunoperoxidase techniques, less than 50% of the highly metastatic MTLn3 tumor cells were stained, whereas approximately 20% of the intermediate metastatic MTF7 and MTLn2 cells and less than 10% of low metastatic MTC and MTPa cells were stained with MAb GP21:56. The cell-to-cell reactivity was heterogeneous and mainly associated with the tumor-cell surface and extracellular matrix.

Animals↗

PubMed1978

Reactivation of nuclei containing lens fiber cells to mitotic growth biochemical and immunochemical analysis.

Nuclei containing lens fiber cells can be reactivated to mitotic growth by transplantation into a suitable culture medium. Reactivated epithelium-like fiber cells are morphologically indistinguishable from cultivated epithelial cells. During reactivation the enzyme pattern of the differentiated fiber cells changes to a pattern closely related to that of cultivated epithelial cells. Moreover dedifferentiation was correlated with an appreciable loss of specific lens proteins (alpha-crystallin). Cytoplasmic shedding and attachment of fiber cells to the substratum are discussed with regard to their role in the reactivation process.

Cell Differentiation↗

PubMed1992

Identification of subgroups of bovine respiratory syncytial virus.

The occurrence of antigenic variation among nine isolates of bovine respiratory syncytial virus (BRSV) was determined by examining their reaction patterns to human respiratory syncytial virus (HRSV) subgroup A and B monoclonal antibodies (MAbs) by enzyme immunoassay and radioimmunoprecipitation with fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using MAbs and polyclonal antisera to HRSV and BRSV. Shared epitopes were demonstrated on four of five structural proteins between BRSV and both subgroups A and B of HRSV. The nine isolates of BRSV showed different patterns of reactivity in enzyme immunoassays with panels of MAbs to HRSV subgroups A and B. Major variations in the molecular weights of the P (phosphoprotein) and F (fusion protein) proteins were demonstrated among the BRSV isolates tested. These results suggest that BRSV belongs to a different antigenic grouping than HRSV and that BRSV is composed of two distinct subgroups.

Animals↗

PubMed1991

A biotin-streptavidin enzyme immunoassay for detection of antibodies to porcine granulosa cell antigens.

A colorimetric solid-phase enzyme immunoassay has been developed which quantifies antibodies to porcine granulosa cell membrane antigens in rabbits immunized with porcine granulosa cells. A cell-free, particulate membrane preparation of porcine granulosa cells was used as coating antigen. A biotinylated second antibody in conjunction with a streptavidin-beta-galactosidase conjugate was utilized to amplify reactivity. The enzyme beta-galactosidase was used due to high background obtained using peroxidase, presumably due to endogenous peroxidase activity of the tissue. Sigmoidal serum dilution curves were obtained with immune rabbit sera indicating that absorbance was related to the concentration of antibodies. Assay activity was reduced by preincubation of immune serum with granulosa cell membranes. Sera from ovariectomized or pre-immune rabbits did not yield any specific binding in the assay. This assay has potential applicability for quantifying antiovarian and antigranulosa cell antibodies in women suspected of having autoimmune premature ovarian failure.

Animals↗

PubMed1987

Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry.

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.

Acid Phosphatase↗

PubMed1985

Dithiocarbamate fungicides decrease histochemical reactivity of cholinesterases in the gut wall of the rat.

The toxicity of dithiocarbamate fungicides is poorly known, especially in the autonomic nervous system. Male Wistar rats were given tetraethylthiuramdisulfide (DSF), maneb, and zineb as a suspension in saline i.p. 200 mg/kg daily for 4 d. Rats received a similar dose of ethylenethiourea (ETU) as a solution in saline i.p., and the control rats received a corresponding volume of saline i.p. After decapitation, pieces of ileum were taken, and the enzyme histochemical reactivity for the acetylcholinesterase (AChE) and for the nonspecific cholinesterase (nsChE) in the ilea of the treated and control animals was studied. The reaction intensities in the samples were estimated by eye using a scale from 0 (no reaction product) to 3+ (maximal reaction). DSF was used as an internal standard. Controls showed the maximal reaction for the AChE and for the nsChE. Maneb, ETU and zineb showed consistent decrease in the reactivity for the AChE (+ +) and for the nsChE (+ +). The decrease by DSF of the reactions for both the AChE (+) as well as for the nsChE(+) was even more pronounced indicating a possible nerve damage to the cholinergic innervation of rat intestine. Kinetic differences may explain the divergent effects between the agents studied.

Animals↗

PubMed1991

Studies on dilution inactivation of sheep liver pyruvate carboxylase.

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.

Acetyl Coenzyme A↗