Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enzyme Reactivators”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources
PubMed1994

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

Amino Acid Sequence↗
PubMed1993

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Adult↗
PubMed1986

Role of Escherichia coli alpha-hemolysin and bacterial adherence in infection: requirement for release of inflammatory mediators from granulocytes and mast cells.

We investigated the role of bacterial mannose-resistant fimbriation of S fimbriae (Fim), mannose-resistant hemagglutination (S-Mrh), and hemolysin (Hly) production by an Escherichia coli parent and genetically cloned strains as regards their effect on histamine release from rat mast cells and generation of the chemiluminescence response, leukotriene, and enzyme release from human polymorphonuclear granulocytes. These mediators are involved in the induction of inflammatory disease processes and lead, e.g., to the enhancement of vascular permeability, chemotaxis, aggregation of granulocytes (leukotriene B4), lysosomal enzyme release, and smooth-muscle contraction (leukotrienes C4, D4, and E4). The content of azurophilic and specific granules in polymorphonuclear granulocytes consists of highly reactive enzymes which amplify inflammatory reactions. Washed bacteria (E. coli 764 Hly+/-, E. coli 21085 Hly+/- Fim+/- Mrh+/-), as well as their culture supernatants, were analyzed at various times during their growth cycle. No differences exist between parent and cloned or mutant strains with respect to their outer membrane proteins and lipopolysaccharide pattern. Washed bacteria [E. coli 764 and 21085(pANN202-312)] which produced hemolysin, unlike Hly- strains, induced high levels of histamine release from rat mast cells and led to a significant chemiluminescence response and enzyme and leukotriene release from human polymorphonuclear granulocytes. Bacterial culture supernatants from Hly+ and secreting strains showed similar results with the exception of E. coli 21085(pANN202-312), which is a hemolysin-producing but not a secretory strain. Our data suggest a potent role for hemolysin as a stimulus for noncytotoxic mediator release from various cells. Furthermore, we showed that the presence of Fim and S Mrh potentiates mediator release. The simultaneous presence of Mrh and Fim [E. coli 535/21(pANN801-4)] increased mediator release compared with Mrh+ Fim- strains [E. coli 536/21(pANN801-1)]. E. coli 536/21 (Msh- Mrh- Fim- Hly-) did not induce mediator release.

Arachidonic Acid↗
PubMed1991

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 4. Effect of various side-chain substituents on therapeutic activity against anticholinesterase intoxication.

A series of quaternary salt derivatives of 2-[(hydroxyimino)methyl]-1-methylimidazole incorporating various side chains bearing ether, silyl, nitrile, ester, halogen, nitro, sulfone, amino, or aminosulfonyl substituents was prepared and evaluated in vivo for the treatment of anticholinesterase intoxication. Test results in the mouse revealed that the type and location of the side-chain substituent both have a significant influence on the toxicity and antidotal effectiveness of the compounds. Some of the more active examples represent the most potent therapeutics to date against intoxication by the powerful cholinesterase inhibitors soman and tabun. Significantly, the antidotal effectiveness of the compounds was not dependent on the inhibiting agent nor was there any correlation between in vivo efficacy and in vitro reactivation of ethyl (4-nitrophenyl)methylphosphonate inhibited human acetylcholinesterase. These observation suggested that the main mode of antidotal protection by the compounds is something other than enzyme reactivation.

Acetylcholinesterase↗
PubMed2002

Transglutamines and endocrine system (minireview).

Transglutaminases catalyze the posttranslation modification of proteins by catalyzing Ca2+ dependent acyl-transfer reaction resulting in the formation of new g-amide bonds between g-carboxamide groups of peptide-bound glutamine residues and various primary amines. Such glutamine residue serves as acyl-donor and the most common acyl-acceptors are e-amino groups of peptide-bound lysine residues or primary amino groups of some naturally occurring polyamines, like putrescine or spermidine. The active site of cysteine reacts first with the g-carboxamide group of glutamine residue to form the acyl-enzyme intermediate under the release of ammonia. In the second step, the complex reacts with a primary amine to form an isopeptide bond and liberate the reactivated enzyme. The presence of transglutaminases has been observed in various endocrine glands such as human pituitary, which was investigated by immunohistochemical methods using specific antibodies. A significant increase in the expression and activity of tissue transglutaminase was observed during involution of thymus. In the genital tract of the male rat two different forms of the enzyme transglutaminase could be identified and characterized. the presence of p53 and tissues transglutaminase gene expressions in human normal and pathologic adrenal tissues. The Ca2+-responsive enzyme transglutaminase, which catalyzes the cross-bridging of proteins, was found in pancreatic islet cells.

Animals↗
PubMed1984

Glyceraldehyde 3-phosphate dehydrogenase activity in rat and human lenses and the fate of enzyme molecules in the aging lens.

The specific activity of the enzyme glyceraldehyde 3-phosphate dehydrogenase declines as a function of age. Immunotitration with monospecific antibodies demonstrated that with age there is an increase of catalytically defective, but antigenically reactive enzyme molecules in the lens. Antiserum, produced against denatured enzyme, removed the inactive molecules from lens homogenates, without effects on the levels of enzyme activity. These studies suggest that inactive enzyme molecules in aging lenses are totally devoid of catalytic activity, and are at least partially denatured.

Adult↗
PubMed2004

Ectopic Agouti protein overexpression increases stimulated corticosterone production without effect on adenylate cyclase activity in mouse adrenal cells.

OBJECTIVE: The antagonism of Agouti protein (AP) and Agouti-related protein on melanocortin receptors suggests an inhibitory role in the regulation of steroidogenesis. However, we have previously demonstrated that ectopic AP overexpression increased restraint-induced corticosterone release and adrenal reactivity to ACTH in mice. A high steroidogenic response to ACTH may be a consequence of a stimulatory AP action on the adenylate cyclase (AC) and/or intracellular steroidogenic enzymes. The aim of the present study was to estimate the effect of ectopic AP overexpression on the activity of AC and steroidogenic intracellular enzymes. METHODS: ACTH and forskolin were used for AC stimulation, and dibutyryl cAMP and progesterone were used for stimulation of intracellular steroidogenic enzymes in isolated adrenal cells in male C57Bl/6J mice of two Agouti genotypes: A(y)/a (ectopic AP overexpression) and a/a (absence of AP in all tissues). RESULTS: ACTH and forskolin increased cAMP accumulation to the same extent in both A(y)/a and a/a mouse adrenal cells (P<0.001; ANOVA), but resulted in higher corticosterone production in A(y)/a mice (P<0.001 for ACTH and P<0.01 for forskolin; ANOVA). Dibutyryl cAMP- and progesterone-induced corticosterone production was higher in A(y)/a mice than in a/a mice (P<0.001 for dibutyryl cAMP and P<0.01 for progesterone; ANOVA). CONCLUSIONS: Ectopic AP overexpression increased stimulated corticosterone production and intracellular steroidogenic enzyme reactivity to cAMP without an effect on AC activity.

Adenylyl Cyclases↗
PubMed1989

[The red light of the helium-neon laser reactivates superoxide dismutase].

The effect of low-energy helium-neon laser (HNL) on enzymatic activity, absorbtion spectra and electron paramagnetic resonance (EPR) signals of superoxide dismutase (SOD) from bovine erythrocytes in acid medium were investigated. It was found that incubation during 2 hours at pH 5.9 led to eventually complete inactivation of the enzyme. The subsequent illumination of inactivated SOD by HNL brought about the enzyme reactivation. Both absorption and EPR-spectra were changed after incubation at pH 5.9. These changes may be attributed to protonation of histidine residue in the enzyme active site. After laser irradiation both absorption and EPR spectra were restored to those typical of native enzyme at pH 8.2. In a model system, copper-histidine complex, absorption maximum was shifted from 632-633 nm at pH 5.8 to 639-640 nm at pH 8.5-9.0. The similar long-wave length shift of the maximum was observed after illumination by HNL at pH 5.8. It may be postulated that the photoreactivation of SOD consists essentially in deprotonation of His-61 residue in the enzyme active site and subsequent recovery of imidazole bridge between copper and zinc which had been destroyed at low pH. Since many other enzymes possess similar copper-histidine structures in their active sites, one may expect diverse effects of red (laser) light on the enzyme activity.

Animals↗
PubMed1990

Kinetic analysis of regeneration by dilution of a covalently modified protein.

An analysis of regeneration by dilution of a covalently modified protein is presented. It is shown that, when protein regeneration is realized through the intermediacy of a protein-modifying agent adsorptive complex, the reaction is described by a summation of two exponential functions of reaction time plus a constant-term equation. The conditions whereby this equation reduces to a single-exponential equation are delineated. It is shown that, when protein regeneration is described by a single-exponential function of reaction time, the first-order protein-regeneration rate constant is a function of modifying-agent concentration and also of the microscopic reaction rate constants. Accordingly, the protein-modifying agent dissociation constant (Ki), as well as the protein-covalent-modification and -regeneration, rate constants (k+2 and K-2), may be determined by an analysis of dilution-induced protein-regeneration (or enzyme-reactivation) data obtained at different dilutions of the covalently modified protein-modifying agent preparation.

Adsorption↗
PubMed1991

Purification and characterization of a membrane-bound acid phosphatase of Leishmania mexicana.

As defined by the reaction with monoclonal antibodies, Leishmania mexicana promastigotes contain two acid phosphatases which together comprise about 90% of the cellular activity. A first enzyme recognized by monoclonal antibody AP4 is largely membrane-bound. The protein has an apparent molecular weight of 70,000-72,000, carries about seven N-linked glycan chains and is present in approximately 16,000 copies per cell. The protein is also expressed in the amastigote stage. A second enzyme reactive with monoclonal antibody AP3, that also recognizes lipophosphoglycan and a secreted acid phosphatase, is mainly found in the soluble fraction of promastigote lysates. It is suggested that this enzyme is the precursor of the secreted protein. The N-terminal sequences of the phosphatase recognized by AP4 and the secreted enzyme are similar but not identical. AP4 does not cross-react with phosphatase activity of Leishmania major or Leishmania donovani promastigotes, while AP3 recognizes part of the cellular and all of the secreted phosphatase activity of L. donovani promastigotes but not that of L. major which does not release an acid phosphatase into the culture medium.

Acid Phosphatase↗
PubMed2004

Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations.

The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

Bacterial Proteins↗
PubMed1977

Role of Phenolic Inhibitors in Peroxidase-mediated Degradation of Indole-3-acetic Acid.

7-Hydroxy-2,3-dihydrobenzofuran derivatives, metabolites of a carbamate insecticide carbofuran, and five other phenolic inhibitors of indoleacetic acid (IAA) oxidase interfered with IAA-induced spectral change in the Soret band of horseradish peroxidase (HRP). The onset of IAA degradation required transformed HRP intermediates. The inhibitors, when added before IAA, protected HRP from reacting with IAA, thus preventing formation of highly reactive enzyme intermediates, and consequently, IAA degradation. When added after IAA, the inhibitors quickly reversed the IAA-induced spectral change of HRP and inhibited further IAA degradation.The phenolic inhibitors differed in stability and reactivity. 7-Hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, 3,7-dihydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, catechol, protocatechuic acid, caffeic acid, ferulic acid, and scopoletin belonged to one group which produced only a temporary inhibition to IAA-induced spectral change of HRP and IAA degradation since the inhibitors were metabolized in the reaction. The length of the lag was dependent on the IAA, inhibitor, and enzyme concentrations. 3-Keto-7-hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran and 3-keto-carbofuran belonged to the other group which produced a persistent inhibition.Degradation of IAA required both the heme group and apoprotein of HRP. Reconstituted enzyme from bovine hemin and apoprotein or HRP after unfolding by urea or guanidine treatment were inhibited by the inhibitors in a way similar to the native HRP. The inhibition was reversible by higher concentrations of IAA, but the plot of 1/v versus 1/s and 1/v versus i were curvilinear, reflecting the complex nature of a competitive inhibition.

Journal Article↗
PubMed1968

Effect of glutathione on ribonuclease.

GSH, but not GSSG, inhibits the reactivation by phosphate ion of ribonuclease activity inactivated by urea or guanidine. The effects of GSH are rather slow and pretreatment of ribonuclease with urea is a requisite for the inhibitory action of GSH on enzyme reactivation. GSH is more effective in urea than in guanidine and its action is greatly enhanced by EDTA. An optimum pH of about 9.0 was found for the inhibitory effect of GSH. Titration of the thiol groups formed after inactivation of ribonuclease by GSH strongly suggests that the reduction of only one disulphide linkage is involved. The reduction of this bond is sufficient to completely abolish the enzymic activity.

Chemical Phenomena↗
PubMed2002

Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants.

To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals. Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits. They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide. Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms. However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride. When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB. These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C. However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained. The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.

Citrate (si)-Synthase↗
PubMed1992

Copper as a cofactor and regulator of copper,zinc superoxide dismutase.

Copper,zinc superoxide dismutase (CuZnSOD), an antioxidant enzyme, is unique in requiring two essential metals for catalytic function. Yet, only one, copper, seems to regulate the expression of functional activity. Restricting dietary copper quickly impairs catalytic functioning of CuZnSOD in numerous tissues. Diets supplemented with copper or small amounts of CuCl2 administered intraperitoneally restore the enzyme activity in animals deprived of copper. Thus, CuZnSOD has been considered a good marker of copper status. A metal-free (apo) form of CuZnSOD could exist in tissues at all times, but especially when an animal is deprived of copper. Restoring CuZnSOD activity with copper permits elucidation of the pathway of copper incorporation into the enzyme. Ceruloplasmin and albumin transport copper to the enzyme in vitro. K562 cells, a human erythroleukemic cell line, can extract copper from ceruloplasmin and incorporate it into CuZnSOD. Ascorbic acid stimulates the transfer of 67Cu transfer from ceruloplasmin to the cells, and somewhat unexpectedly, appears to restrict the amount of transferred copper that becomes bound to the enzyme. Reactivation of CuZnSOD in healthy individuals has the potential of being a useful tool for assessing copper status. This approach has merit, but one must consider that the levels of apo-enzyme that prevail in tissue could be influenced by other metals.

Animals↗
PubMed1975

Paramagnetic and fluorescent probes attached to "buried" sulfhydryl groups in human carbonic anhydrases. Application to inhibitor binding, denaturation and refolding.

1. The single -SH groups in the human carbonic anhydrases B and C have been modified under denaturing conditions. The modified enzymes recover catalytic activity after dilution of the denaturing medium with buffer. By this method a spin label and a fluorescent probe were specifically introduced into the molecules. 2. The modified and reactivated enzymes have similar kinetic properties, inhibitor-binding constants, circular dichroism spectra, and stabilities towards guanidine hydrochloride as the native enzymes. However, the esterase activity of the modified C enzyme is reduced to about 50%. 3. The spectra associated with the probes are insensitive to inhibitor binding in case of the B enzyme, whereas changes of electron paramagnetic resonance spectrum and fluorescence intensity respectively, were observed for the probe-containing C enzymes. The cysteines are located in different parts of the tertiary structures of the homologous B and C enzymes, and these observations suggest that small conformational changes accompanying inhibitor binding are localized to regions of the molecules near the active-site cavity. 4. During denaturation of the spin-labeled B enzyme in 1.7 M guanidine hydrochloride a transient mobilization of the probe occurs, but the mobility is ultimately reduced to a low level. This observation supports previous evidence that denaturation under these conditions, in or near the transition region, mainly yields incorrectly folded molecules rather than stable intermediates between native and fully denatured molecules. 5. During refolding of fully denatured, spin-labeled B and C enzymes the mobility of the probe is drastically reduced within less than 0.1 s after dilution. This would reflect a very short lifetime of the randomly coiled state under these conditions.

Binding Sites↗
PubMed2001

Carboxylesterase: specificity and spontaneous reactivation of an endogenous scavenger for organophosphorus compounds.

The ability of carboxylesterase (CaE) to act as a bioscavenger to provide protection against organophosphorus (OP) compounds has been demonstrated in several animal models. To further evaluate the effectiveness of CaE as a bioscavenger, the specificity and stoichiometry of the detoxication of OP compounds by rat plasma CaE were examined. The specificity of CaE was evaluated by determining the bimolecular rate constants for inhibition (k(i)) of CaE by a variety of OP compounds. CaE exhibited a broad specificity for neutral OP compounds with k(i) > 10(6) M(-1) x min(-1) for paraoxon, sarin, soman, diisopropyl fluorophosphate, and diphenyl p-nitrophenyl phosphinate. CaE exhibited poor reactivity (k(i) < 10(4) M(-1) x min(-1)) with cationic OP compounds, such as echothiophate, VX, and iso-OMPA. The stoichiometry of CaE detoxication of OP compounds was evaluated by determining the rates of enzyme reactivation and ageing of OP-inhibited CaE. CaE exhibited no ageing after inhibition by any of the OP compounds, including soman. However, OP-inhibited CaE did exhibit spontaneous reactivation with reactivation rates that decreased as the size of the OP increased (i.e., VX > sarin > soman). The pH dependence of the spontaneous reactivation of sarin-inhibited CaE suggested that its reactivation was dependent on an amino acid residue with a pK(a) of 6.1, which is probably a histidine that is highly conserved in CaE but not in other esterases.

Carboxylesterase↗