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Results for “Enzyme Reactivators”

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PubMed1991

[Changes in intra-renal scavenging enzymes activities of the reactive oxygen species in experimental glomerulonephritis and nephrosis in rats].

The purpose of this study was to investigate the changes in scavenging enzymes activities of reactive oxygen species (ROS) in experimental nephritis and nephrosis. Anti-GBM nephritis was induced in rat by injecting 0.75 ml of the anti-GBM serum. Intrarenal SOD-like activity in nephritic rats was significantly decreased from the 5th day after i.v. injection of antiserum to the 10th day. Although catalase and glutathione peroxidase activities in nephritic rat were increased on 3hr to 6hr after i.v. injection of antiserum, the both activities were gradually decreased on 24hr onward. The decrease in ROS scavenging enzymes activities paralleled with the urinary protein excretion and the elevation of plasma lipid peroxide. Moreover, catalase, administered (4,600 U/hr) by osmotic minipump prevented the urinary protein excretion by about 60%. In accelerated passive Heymann nephritis and puromycin aminonucleoside nephrosis in rats, the activities of scavenging enzymes decreased in the course of experimental periods. It was concluded that the scavenging enzymes activities of ROS increased at beginning of heterologous phase and decreased at the end of this phase in anti-GBM nephritis, and the activities in the other models of experimental nephritis also decreased during experimental periods.

Animals↗

PubMed1983

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli.

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.

Chlorides↗

PubMed1982

Concanavalin A alters the turnover rate of tyrosine aminotransferase in cultured hepatoma cells.

Concanavalin A added to monolayer cultures of Reuber H-35 hepatoma cells caused a rapid inactivation of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, E.C. 2.6.1.5) and loss of reactivity with antibody against the native, dimeric enzyme. Analysis of treated cells with an antibody raised against carboxymethylated, denatured enzyme showed that the inactivated enzyme was reactive with this reagent, which does not react with the native enzyme. Subsequent addition of alpha-methyl-D-mannopyranoside to remove concanavalin A restored both enzyme activity and reactivity to antibody against native enzyme. After long-term treatment with concanavalin A, the restored enzyme levels were significantly higher than in controls treated with the sugar but not the lectin. Analysis of the turnover of the enzyme by two methods revealed that the rate of its degradation is reduced about 2-fold in concanavalin A-treated cells. Treatment with H-35 cells with concanavalin A thus effects an alteration in conformation of tyrosine aminotransferase, rendering it somewhat less sensitive to intracellular degradation.

Animals↗

PubMed1987

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects on the sulfur-free and sulfur-containing catalytic intermediates.

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave Km (Ks) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reversed by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.

Anilino Naphthalenesulfonates↗

PubMed1995

C-terminal domain of human pancreatic lipase is required for stability and maximal activity but not colipase reactivation.

Fungal lipases and human pancreatic lipase (hPL) share a common tertiary structure termed the alpha/beta hydrolase fold. In contrast, the region C-terminal to the common tertiary structure does not share any common structural features with fungal lipases, leading to the hypothesis that the divergent C-terminal domain confers specific properties to hPL. To study the role of the C-terminal domain in hPL function, we made substitution and deletion mutations in the C-terminal domain. The mutant proteins were expressed in transfected COS-1 cells and the secreted proteins were analyzed by immunoblot and for lipase activity. Substitution mutants in multiple lysine residues, in aspartate 390, or in tyrosine 404 did not affect secretion or lipase activity of the mutants. Significantly, the mutants still required colipase for maximal activity. Deletion of the C-terminal domain decreased the amount of truncated, mutant protein in the medium of transfected cells and decreased the specific activity of the mutants. Still, maximal activity required colipase, indicating that the deletion mutants interacted with colipase. Interfacial binding of the truncated deletion mutants was decreased relative to wild-type hPL. The newly synthesized deletion mutants were not as efficiently secreted from the transfected cells as wild-type hPL, and the mutant proteins that appeared in the medium were less stable than the wild-type hPL. These findings suggest that the C-terminal domain is required for proper folding or processing of hPL, confers stability, and increases activity, but is not absolutely required for colipase reactivation of the bile salt-inhibited enzyme.

Amino Acid Sequence↗

PubMed1986

The enzyme rhodanese can be reactivated after denaturation in guanidinium chloride.

For the first time, the enzyme rhodanese has been refolded after denaturation in guanidinium chloride (GdmHCl). Renaturation was by either (a) direct dilution into the assay, (b) intermediate dilution into buffer, or (c) dialysis followed by concentration and centrifugation. Method (c) preferentially retained active enzyme whose specific activity was 1140 IU/mg, which fell to 898 IU/mg after 6 days. The specific activity of native enzyme is 710 IU/mg. Progress curves were linear for the dialyzed enzyme, and kinetic analysis showed it had the same Km for thiosulfate as the native enzyme, but apparently displayed a higher turnover number. Progress curves for denatured enzyme directly diluted into assay mix showed as many as three phases: a lag during which no product formed; a first order reactivation; and an apparently linear steady state. An induction period was determined by extrapolating the steady-state line to the time axis. The percent reactivation fell to 7% (t1/2 = 10 min) as the time increased between GdmHCl dilution and the start of the assay, independent of the presence of thiosulfate. The induction period, which decreased to zero as the incubation time increased, was retained in the presence of thiosulfate. There were no observable differences between native and renatured protein by electrophoresis or fluorescence spectroscopy. Previous reports of some refolding of urea-denatured rhodanese (Stellwagen, E. (1979) J. Mol. Biol. 135, 217-229) were confirmed, extended, and compared with results using GdmHCl. A working hypothesis is that rhodanese refolding involves intermediates that partition into active and inactive products. These intermediates may result from nucleation of the two rhodanese domains, which exposes hydrophobic surfaces that become the interdomain interface in the correctly folded protein.

Animals↗

PubMed1999

[Expression of reactive oxygen-related enzymes in human umbilical vein endothelial cells (HUVEC) cultured with high concentrations of glucose].

We examined the levels of reactive oxygen-related enzymes in human umbilical vein endothelial cells cultured with high concentrations of glucose, in vitro. From the results, elevated levels of catalase mRNA and its protein were exhibited in the presence of higher glucose. In addition, the message level of p22-phox as the active center of NADPH oxidase, was slightly increased. Taken together, the endothelial injury induced by diabetes may associated to the elevated level of O2- production. However, the level of catalase as .OH scavenger was mainly increased, cooperatively.

Cells, Cultured↗

PubMed1998

The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis.

Phenobarbital and clofibrate, two non-genotoxic carcinogens, have been investigated regarding the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis in primary cultures of rat hepatocytes. Low toxicity concentrations, 200 and 100 microg/ml for phenobarbital and clofibrate respectively, were used to examine their effect on spontaneous or transforming growth factor beta1 (TGFbeta1)-induced apoptosis and on the expression of antioxidant defence enzymes (superoxide dismutases and catalase). The increased incidence of apoptotic nuclei was visualized in TGFbeta1-treated cultures with the fluorescent dye Hoechst 33258 and was quantified under all experimental conditions by measurement of the hypodiploid peak in DNA histograms obtained by flow cytometry. Both substances, when added separately to hepatocyte cultures and incubated for 24 and 48 h, significantly diminished spontaneous apoptosis and exhibited a slight suppression of TGFbeta1-induced apoptosis. Endogenous peroxide production by hepatocytes increased with TGFbeta1, phenobarbital or clofibrate and the increase was greater with phenobarbital and in the presence of TGFbeta1 with both drugs. Gene expression of catalase and Mn- and Cu,Zn superoxide dismutases (SOD) was evaluated by northern blot analysis of hepatocytes incubated in the presence of phenobarbital or clofibrate with or without TGFbeta1 and the following differences were detected: phenobarbital induced a significant decrease in both dismutases (to 56%, P < 0.05, and 55%, P < 0.05, for Mn- and Cu,Zn-SOD respectively) and a 2-fold increase (P < 0.01) in catalase; clofibrate induced a slight decrease in both SODs and a 4-fold increase (P < 0.05) in catalase; TGFbeta1 significantly decreased to 37% (P < 0.05) expression of catalase while not significantly affecting expression of both SODs. We conclude that inhibition of spontaneous apoptosis induced by either phenobarbital or clofibrate is accompanied by increases in the endogenous levels of peroxides and by significant induction of catalase gene expression. Furthermore, the lack of effect of both compounds on TGFbeta1-induced apoptosis could be a consequence of the inability of these two compounds to counteract the depressing effect of TGFbeta1 on expression of catalase.

Animals↗

PubMed1981

An enzymatic assay for available zinc in plasma and serum.

A convenient enzymatic assay for available zinc in plasma and serum was developed. The assay is based upon the reactivation of the zinc metalloenzyme alkaline phosphatase which had been previously inactivated by nitrilotriacetic acid. The enzymatic assay was applied to serum samples from human subjects in a study of zinc utilization in pregnancy and to plasma samples from rats in a zinc deficiency study. Most plasma samples with low zinc levels (less than 70 microgram/dl) reactivated the enzyme to less than 30% of control activity. Most samples with normal zinc levels (70-120 microgram/dl) reactivated the enzyme 30-65%. Samples with elevated zinc levels (greater than 120 microgram/dl) reactivated the enzyme over 70%. The assay is quick, convenient, and in principle measures functionally available zinc.

Alkaline Phosphatase↗

PubMed1986

Kinetics of protein-modification reactions. Determination of the fractional concentration of enzyme protein groups, or group reactivities, essential for catalytic function.

A mathematical treatment of modification-induced enzyme protein inactivation is presented, and it is shown that, at initial reaction conditions, the ratio of the first derivative of the equation describing enzyme activity loss to the first derivative of the equation describing protein groups modification is equal to the fractional concentration of enzyme protein reactive groups, or group reactivities, essential for catalytic function.

Catalysis↗

PubMed1985

Reactivation kinetics of diethylphosphoryl acetylcholine esterase.

The kinetics of reactivation of diethylphosphorylated acetylcholine esterase by pyridine-2-aldoxime methochloride has been studied using the approach of following the course of the hydrolysis of acetylcholine during the reactivation of the phosphorylated enzyme by the reactivator [Tsou, C.-L. (1965) Acta Biochem. Biophys. Sin. 5, 398-417]. Equations are derived based on the scheme of the formation of a complex between the phosphorylated enzyme and the reactivator and the rate of dissociation of this complex is not necessarily faster than the dephosphorylation and regeneration of the active enzyme. The regenerated enzyme then reacts with the substrate through an acetyl-enzyme intermediate as generally depicted. The equation obtained for product formation during the course of reactivation contains two exponential terms and this is in accord with the experimentally observed biphasic reaction. By making the assumption that the dissociation of the phosphorylated enzyme-reactivator complex is much faster than the dephosphorylation reaction, the above equation can be simplified to a form containing only one exponential term. By following the course of the reactivation reaction with the conventional approach of taking aliquots and assaying for enzyme activity recovery, it would appear likely that one would miss the initial stage of this biphasic reaction.

Acetylcholinesterase↗

PubMed1987

Sympathetic nervous activity and noradrenaline reactivity during angiotensin converting enzyme inhibition.

The effect of the angiotensin converting enzyme inhibitor ramipril on catecholamine disposition and noradrenaline reactivity was studied in normotensive volunteers. In the first study 5 mg of ramipril or placebo was given 3 times at 12-hour intervals in a randomized, double-blind, cross-over manner (n = 10). In the second study, ramipril 10 mg daily was given for 2 weeks (n = 6). Noradrenaline reactivity increased significantly (p less than 0.05) both in short- and long-term application, while blood pressure decreased (p less than 0.01). Sulfoconjugated plasma noradrenaline decreased significantly (p less than 0.05) possibly indicating a decrease in sympathetic tone. These findings suggest that a decrease in sympathetic tone could contribute to the blood pressure-lowering effect of ramipril, whereas the increase in noradrenaline reactivity is probably a consequence of the primary change in sympathetic activity.

Adult↗

PubMed2002

Use of enzyme treatment to enhance reactivity of HLA and platelet-specific antibodies in solid-phase RBC adherence assays.

BACKGROUND: Solid-phase RBC adherence (SPRCA) assays (Immucor) detect HLA and/or platelet-specific antibodies. Pretreatment of reagent platelets with enzymes was investigated to determine whether the sensitivity of the assay could be increased. STUDY DESIGN AND METHODS: SPRCA testing, following the manufacturer's directions, was performed on 51 sera from patients with either a positive SPRCA antibody screen, suboptimal clinical responses to platelet transfusions, and/or suspected immune thrombocytopenic purpura; testing was also performed following pretreatment of the reagent platelets with bromelin, papain, or ficin. Sera from 23 patients having negative routine SPRCA antibody screens and good clinical responses to transfusion were tested as controls. Lymphocytotoxic antibody testing was also performed on selected samples. The effectiveness of enzyme treatment was judged by the increase in the proportion of reagent platelets reacting with the sample and the observed reaction strengths. RESULTS: Pretreatments of reagent platelets with all three enzymes increased the reactivity of known antibodies and detected some HLA and platelet-specific antibodies that had not reacted in routine testing. The clinical significance of the antibody specificities detected only after enzyme pretreatment was verified by a correlation with results from transfusing antigen-negative units. Only occasional false-positive results after enzyme pretreatment were observed. CONCLUSIONS: The use of enzyme pretreatment of SPRCA screening strips can provide information that is useful in selecting appropriate units for transfusion.

Antibody Specificity↗

PubMed1994

Oxidation therapy: the use of a reactive oxygen species-generating enzyme system for tumour treatment.

Oxygen radicals induce cytotoxicity via a variety of mechanisms, including DNA damage, lipid peroxidation and protein oxidation. Here, we explore the use of a polyethylene glycol (PEG)-stabilised enzyme capable of producing reactive oxygen species (ROS), glucose oxidase (GO), for the purpose of harnessing the cytotoxic potential of ROS for treating solid tumours. PEG-GO (200 U), administered by two intratumoral injections 3 h apart, produced a significant growth delay in subcutaneous rat 9L gliomas as compared with control animals receiving heat-denatured PEG-GO. Rats were protected from systemic toxicity by subsequent i.v. administration of PEG-superoxide dismutase (PEG-SOD) and PEG-catalase. In vivo tumour metabolic changes, monitored using 31P magnetic resonance spectroscopy (31P-MRS) 6 h following initial administration of PEG-GO, revealed a 96 +/- 2% reduction in the ATP/Pi ratio and a 0.72 +/- 0.10 unit decline in intracellular pH. A 3-fold sensitisation of 9L glioma cells in vitro to hydrogen peroxide could be achieved by a 24 h preincubation with buthionine sulphoximine (BSO). This study suggests that oxidation therapy, the use of an intratumoral ROS-generating enzyme system for the treatment of solid tumours, is a promising area which warrants further exploration.

Animals↗

PubMed1989

IgM to human cytomegalovirus: comparison of two enzyme immunoassays and IgM reactivity to viral polypeptides detected by immunoblotting.

IgM detection by enzyme-linked immunosorbent assay (ELISA) is the most used method of establishing a cytomegalovirus (CMV) infection during pregnancy. In this paper, we discuss the results obtained assaying 1,000 sera from pregnant women by two ELISA kits: a traditional indirect ELISA and a more recent IgM-capture ELISA. All the sera that gave a positive ELISA value with one or both kits were further tested by immunoblotting (IB) to establish which CMV polypeptides were detected by IgM antibodies. From the results obtained, IgM-capture ELISA seems less sensitive than indirect ELISA, but correlates better with serological evidence of active CMV infections as judged by typical IB profiles.

Antibodies, Viral↗

PubMed1995

Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes.

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.

Adenosine Triphosphatases↗

PubMed1984

Pseudocooperative effects in reactivation of membrane-bound enzymes with phospholipids.

The kinetic mechanism for reactivation of membrane-bound enzymes by lipids is analyzed on the basis of multiple equilibriums between the enzyme (which is assumed to contain n identical, non-interacting binding sites) and the lipid. The rate equations derived when only the fully occupied enzyme species ELn (or ELn and the next most highly occupied species ELn-1, ELn-2,..., ELn-i) is catalytically active can fully account for the apparent positive cooperativity observed in the plots of enzyme activity as a function of phospholipid concentration. A general equation for the cases in which more than one lipid species are simultaneously present in the reaction medium is presented which allows to test whether the binding sites are indeed non-interacting, as well as whether the active species have the same catalytic constant (kcat), regardless the nature of the lipid bound to the binding sites. This analysis demonstrates that, in addition to apparent positive cooperativity, more complex curves resembling mixed cooperativity may be obtained with simple systems when interaction between protein subunits (i.e., association-dissociation equilibriums) are present. Finally, some theoretical problems and pitfalls in the interpretation of the experimental results will be discussed.

Binding Sites↗

PubMed1979

Modification of lysyl residues of dihydrofolate reductase with 2,4-pentanedione.

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) from an amethopterin-resistant strain of Lactobacillus casei was inactivated by 2,4-pentanedione. The inactivation appears to be due to the specific interaction of 2,4-pentanedione with lysyl residues. Inactivation is concomitant with with the modification of three lysyl residues. Both NADPH and dihydrofolate protect the enzyme against inactivation, suggesting that the critical residue(s) lies at or near their binding sites. Unlike native dihydrofolate reductase, 2,4-pentanedione-modified enzyme does not form binary complexes with either NADPH, dihydrofolate or amethopterin which are stable to gel filtration. Treatment of the modified enzyme with nucleophilic reagents such as hydroxylamine, failed to promote reactivation of the enzyme. Reactivation was achieved following gel filtration at pH 6.0 and was found to be dependent on the degree to which the enzyme was inactivated.

Arginine↗