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Results for “Enzyme Reactivators”

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PubMed2002

Regulation of catalase: inhibition by peroxynitrite and reactivation by reduced glutathione and glutathione S-transferase.

The regulation of stable catalase from Aspergillus niger was investigated. The preincubation of catalase with peroxynitrite (PN) resulted in a significant decrease in the production of O2, while the subsequent incubation with reduced glutathione (GSH, 1mM) led to restoration of the enzymatic activity. Western blot analysis revealed not only the increased immunoreactivities of 3-nitrotyrosine and S-nitrosocysteine in a PN-dose-dependent manner, but also conversely decreased immunoreactivity of 3-nitrotyrosine by the subsequent preincubation of catalase with GSH (1mM)/glutathione S-transferase (GST). The inhibition of the catalase after PN-treatment may be due to conformational changes of the enzyme via tyrosine-nitration/cysteine-nitrosation and the binding of active nitrogen/oxygen species to the Fe3+-protoporphyrin groups of the enzyme. The reverse of these processes to restore enzymatic activity by GSH/GST may be a vital antioxidative mechanism.

Aspergillus niger↗

PubMed2000

Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction.

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldoxime methiodide (2-PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon.

Acetylcholinesterase↗

PubMed1994

Modification of glutamine synthetase in Streptomyces griseus by ADP-ribosylation and adenylylation.

Addition of NH4+ to STreptomyces griseus 2682 cells grown in NO3- containing medium resulted in a rapid decline in glutamine synthetase activity due to covalent modification of the enzyme. The NH4+ promoted inactivation of the enzyme was inhibited by the ADP-ribosyltransferase inhibitor 3-methoxybenzamide. In the presence of ADP-ribosyltransferase activity the purified glutamine synthetase was also inhibited by NAD+ in a concentration-dependent manner. ADP-ribosylation of glutamine synthetase was demonstrated in vitro by showing the incorporation of labeled ADP-ribose from [alpha-32P]NAD+ into glutamine synthetase subunits. Beside ADP-ribosylation, adenylylation of glutamine synthetase was also shown in S. griseus since phosphodiesterase I treatment reactivated the enzyme in crude extracts of NH(4+)-shocked cells. Glutamine synthetase was also inhibited and modified by ATP in crude cellular extracts. These results suggest that in S. griseus 2682 ADP-ribosylation of glutamine synthetase could be an alternative modification to adenylylation to regulate glutamine synthetase activity.

Adenine Phosphoribosyltransferase↗

PubMed1992

In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413).

Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. Böger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) dinitrogenase reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.

Acetylene↗

PubMed1987

Distance estimate of the active center of D-beta-hydroxybutyrate dehydrogenase from the membrane surface.

D-beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) is a membrane-bound, lipid-requiring enzyme which has a reactive sulfhydryl in the vicinity of the active center. The spin-probe-spin-label technique has been used to estimate the distance of separation of the reactive sulfhydryl of D-beta-hydroxybutyrate dehydrogenase from the bilayer surface. The reactive sulfhydryl of the enzyme was derivatized with the maleimide spin-label reagent 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy in the presence of the cofactor NAD+. The derivatized enzyme, inserted (inlaid orientation) into phospholipid vesicles, was titrated with spin probes, either Mn2+ or Gd3+, until the spin-label EPR spectrum was reduced in amplitude to its residual (limiting) value. From this limiting amplitude, the dipolar interaction coefficient was obtained, which is related to the reciprocal of the distance to the sixth power. The radial distances of closest approach of the paramagnetic Mn2+ and Gd3+ ions to the spin-label nitroxide on the enzyme were found to be 18 and 16 A, respectively. These calculated distances were in accord with those determined by comparison with a phosphatidylcholine calibration system having 2,2-dimethyloxazolidinyl-1-oxy spin-labels located at selected positions along the sn-2 fatty acyl chain. Since the distal nitroxide moiety of the maleimide spin-label (17 A from the bilayer surface) is 8 A from the sulfhydryl addition site, the two limiting distances of immersion of the reactive sulfhydryl within the bilayer are 9 and 25 A. The shorter distance is considered more compatible with facile access of the coenzyme to the active site of the enzyme.

Binding Sites↗

PubMed1984

Conversion of low molecular weight renin into the high molecular weight form by Cu2+ in the rat kidney.

The low molecular weight form of renin (M.W. 40000) in the extract of rat kidney cortex was converted into the high molecular weight form (M.W. 60000) in the presence of Cu2+ and this conversion was abolished following pretreatment of the extract with disodium ethylenediaminetetraacetic acid. Fe3+, Fe(CN)6(3-), Cd2+ and Zn2+ had no effects on the reaction of the molecular weight conversion of this enzyme. The reactivity in the formation of high molecular weight renin catalyzed by Cu2+ increased when the extract was previously dialyzed, indicating the presence of substance(s) that interfere with the binding reaction of renin. The high molecular weight renin produced by Cu2+ was stable against dithiothreitol, suggesting that the formation of disulphide bridge(s) is not involved in the reaction of renin with the renin binding substance. We also confirmed that for formation of the high molecular weight renin, Cu2+ must react with both the renin enzyme and the renin binding substance. Our findings provide new evidence related to the mechanism of molecular weight conversion of renin in the rat kidney.

Animals↗

PubMed1984

Inhibition of pig kidney dopa decarboxylase by coenzyme-5-hydroxytryptophan adducts.

The effect of N-(5'-phosphopyridoxyl)-L-5-hydroxytryptophan, N-(5'-phosphopyridoxyl)-D-5-hydroxytryptophan and N-(5'-phosphopyridoxyl)-5-hydroxytryptamine on the reactivation of apoDopa decarboxylase to holoenzyme has been investigated. The different degree of inhibition exerted by these adducts has been interpreted on the basis of a different orientation of the 2 isomers of 5-HTP at the active of Dopa decarboxylase.

Animals↗

PubMed1993

An algorithm for confirming screen reactivity in blood donors in enzyme immunoassays for antibodies to hepatitis C virus.

Three commercial second generation enzyme immunoassays (EIA) and a second generation immunoblot assay (RIBA-II) for detecting antibodies to hepatitis C virus (HCV) were evaluated in a study of confirmatory testing using sera referred by six Regional Blood Transfusion Centres (RTC). Of a total of 490 samples, 203 were negative in the same EIA as that used by the RTC and of these 162 were negative in all three EIAs. The RIBA-II immunoblot test was performed on all samples and 359 were negative, 69 were indeterminate and 62 reactive. We found a close relationship between RIBA-II reactivity, the reactivity of a sample in all three EIAs and the mean of the test/cut-off ratios of all three EIAs performed on that sample. When this was evaluated by PCR for HCV RNA, we found an association between the mean test/cut-off ratio and the probability that an immunoblot reactive or indeterminate sample was PCR positive. A mean ratio of greater than 5.0 in a RIBA-II reactive sample was associated with a 100% probability (16/16 tested) of being PCR positive. These observations should be extended by testing RIBA-II negative samples by PCR so that simplified algorithms for anti-HCV testing can be developed.

Blood Donors↗

PubMed1984

Inhibition of non-specific leukocyte esterase activity. Absence of monocyte esterase activity due to phosphoric and thiophosphoric acid ester intoxication.

In vitro evaluation of the effect of five insecticidal phosphoric and 11 thiophosphoric acid esters on different, non-specific human leukocytes esterases indicated that most of the organic phosphor compounds studied inhibited the activity of neutral alpha-naphthylacetate esterase, alpha-naphthylbutyryl esterase, and naphthol AS acetate esterase, i.e. the monocyte esterases. The extent of inhibition was dose dependent; the inhibiting dose being identical for the various non-specific esterases. Reactivation with Obidoxim was not successful. Monocyte esterase activity in a human survivor of E 605 intoxication was detectable only after serum acetylcholinesterase had returned to normal levels. The organic phosphor compound studied, however, inhibited neither acid alpha-naphthylacetate esterase nor naphthol AS-D chloroacetate esterase activity.

Adolescent↗

PubMed1998

Rapid and irreversible inhibition of creatine kinase by peroxynitrite.

We examined the ability of peroxynitrite and other .NO-derived oxidants to inhibit creatine kinase (CK). Peroxynitrite potently inhibited CK activity and depleted protein thiols. The rate constant for this reaction was 8.85x10(5) M(-1) s(-1). Glutathione did not reactivate CK activity nor did it regenerate protein thiol content. In contrast, glutathione reactivated CK, and regenerated protein thiols, after inhibition by either .NO or oxidized glutathione (GSSG). Peroxynitrite did not irreversibly inhibit CK after it had been treated with GSSG to block protein thiols. We conclude that thiol oxidation is a critical event leading to inactivation of CK by peroxynitrite.

Acetonitriles↗

PubMed1978

[Structure-activity relationship in reactivators of acetylcholinesterase inhibited by phosphoric esters. XII. Hydroxyiminomethylalkylpyridine iodomethylate].

2-Hydroxyiminomethyl-4-alkylpyridine methiodides were prepared and tested for their in vitro reactivating potency on phosphorylated electric eel cholinesterase. They were also tested as in vivo protecting agents: only one of the compounds, 4-methyl-2-hydroxyiminopyridine, offered a greater protection from DFP than 2-PAM, the others had little to no effect.

Acetylcholinesterase↗

PubMed2000

Evidence for a conformational change in subunit III of bovine heart mitochondrial cytochrome c oxidase.

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N'-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl-biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3'-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.

Animals↗

PubMed2006

Inactivation of copper, zinc superoxide dismutase by H2O2 : mechanism of protection.

Cu,Zn SOD is known to be inactivated by HO(2)(-) and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO(2)(-). We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0-9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.

Enzyme Reactivators↗

PubMed1986

Characterization of three aminopeptidases purified from maternal serum.

The biochemical characteristics of aminopeptidase A (EC 3.4.11.7), oxytocinase (EC 3.4.11.3) and alanyl aminopeptidase (EC 3.4.11.2) purified from serum of pregnant women were compared. Aminopeptidase A hydrolysed only acidic amino acid derivatives, whereas oxytocinase and alanyl aminopeptidase had partially overlapping broad substrate specificities. Oxytocinase showed the highest Vmax value with LeuNA but the lowest Km value with ArgNA (Km 0.059 +/- 0.08 mmol/l). Alanyl aminopeptidase hydrolysed AlaNA most rapidly, but showed the highest affinity for LysNA (Km 0.054 +/- 0.006 mmol/l). The enzymes were sensitive to EDTA. Co2+, Ni2+ and Zn2+ were able to reactivate all suppressed enzymes, but Mn2+ reactivated only aminopeptidase A after EDTA inhibition. The alkaline earth metals were activators of aminopeptidase A, while Co2+ activated only alanyl aminopeptidase. This enzyme was the most sensitive to L-amino acids. Acidic amino acids inhibited aminopeptidase A but had no effect on the two other enzymes. Oxytocinase was most sensitive to thermal treatment. Amastatin did not inhibit oxytocinase, whereas aminopeptidase A was more resistant than alanyl aminopeptidase to this effector.

Amino Acids↗

PubMed2005

Selenoprotein expression in Hürthle cell carcinomas and in the human Hürthle cell carcinoma line XTC.UC1.

Hürthle cell carcinomas (HTC) are characterized by mitochondrial amplification and enhanced oxygen metabolism. To clarify if defects in enzymes scavenging reactive oxygen species are involved in the pathogenesis of HTC, we analyzed selenium (Se)-dependent expression of various detoxifying selenoproteins in the HTC cell line XTC.UC1. Glutathione peroxidase and thioredoxin reductase activity was found both in cell lysates and conditioned media of XTC.UC1 cells and was increased by Na(2)SeO(3). Western blot analysis demonstrated the presence of thioredoxin reductase both in cell lysates and conditioned media and of glutathione peroxidase 3 in conditioned media. Type I 5'-deiodinase, another selenoprotein that catalyzes thyroid hormone metabolism, was detectable only in cell lysates by enzyme assay and Western blot, and responded to stimulation by both Na(2)SeO(3) and retinoic acid. A selenoprotein P signal was detected in conditioned media by Western blot, but was not enhanced by Na(2)SeO(3) treatment. In situ hybridization revealed glutathione peroxidase mRNAs in HTC specimen; glutathione peroxidase 3 mRNA levels were reduced. These data suggest adequate expression and Se-dependent regulation of a couple of selenoproteins involved in antioxidant defense and thyroid hormone metabolism in XTC.UC1 cells, so far giving no evidence of a role of these proteins in the pathogenesis of HTCs.

Adenoma, Oxyphilic↗

PubMed1993

Role of oxygen and carbon radicals in hemoglobin oxidation.

We investigated the role of free radicals in hemoglobin (Hb) oxidation and denaturation. To generate free radicals, we used two azocompounds, the hydrophilic 2,2'-azobis(2-amidinopropane hydrochloride and the hydrophobic 2,2'-azobis(2,4-dimethylvaleronitrile) and a drug of the quinone family, phenazine methosulfate. The radical species involved were analyzed by direct EPR and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide, and N-t-butyl-alpha-phenyl-nitrone. The free radicals generated by the azocompounds were carbon radicals and, in the presence of molecular oxygen, peroxyl/alkoxyl radicals. The reaction of phenazine with Hb produced a nitrogen-centered semiquinoid radical detectable by EPR only under N2 and reactive oxygen species (O2-. and H2O2) in the presence of molecular oxygen. Azocompounds oxidized Hb to methemoglobin, hemichromes, and choleglobin while phenazine produced methemoglobin and ferrylhemoglobin. For all three drugs, low oxygen tensions (pO2 = 62 mm Hg) increased the formation of Hb oxidation products, whereas high oxygen tensions (pO2 = 540 mm Hg) reduced Hb oxidation. The formation of irreversible Hb oxidation products (irreversible hemichromes and Hb cross-linking) was observed only with the azocompounds and was reduced at high pO2. Spin traps and thiourea protected Hb from the oxidative damage induced by the azocompounds, whereas enzymes scavenging reactive oxygen species, such as superoxide dismutase and catalase, affected Hb oxidation induced by phenazine and that induced by the hydrophobic azocompound. These results indicate distinct patterns of oxidation and denaturation with each agent. Damage induced by phenazine was dependent on the formation of reactive oxygen species, whereas the damage induced by the azocompounds was due mainly to carbon-centered radicals with some involvement by reactive oxygen species only for the hydrophobic azocompound. The preferential interaction of Hb with drug radicals scavenged by molecular oxygen indicates that this protein may be more reactive under hypoxic conditions and led to the view that a good supply of oxygen can provide an important defense against drug-induced Hb oxidation.

Amidines↗

PubMed1984

Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates.

A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro, or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human beta-factor XIIa (factor XIIa fragment), and activated bovine and human protein C. Kinetic constants (kcat, KM, and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human beta-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140 000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin, and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

[Methods of treating experimental Neguvon poisoning and a comparison of their effects].

The effect of conventional antidotal therapy of oral intoxication due to organophosphorus insecticide Neguvon in male mice is compared to the effect of peritoneal dialysis. As a dialysate, the combination of ACHE reactivator with atropine, distilled water and ACHE preparation has been used. In order to evaluate treatment results, the clinical course of intoxication and the increase of blood ACHE activity inhibited with perorally Neguvon were investigated. The highest increase in activity of inhibited blood ACHE has been stated by the peritoneal dialysis with ACHE preparation. Furthermore, the effects of two dialysates were compared. The first one consisted in ACHE reactivator with atropine, and the second one in ACHE preparation, respectively. The ACHE containing dialysate showed better therapeutical results. The mentioned acetylcholinesterase is stated to be inhibited through the treatment with 50% of Neguvon dosage applied to the experimental animals. Thus the actual dose of Neguvon inducing proper intoxication was reduced to its half amount.

Acetylcholinesterase↗