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 253 records · Page 14Linked to original sources
PubMed1990

Quantitative cytochemistry of lysosomal structures in rat incisor maturation enamel organ.

Trimetaphosphatase was used as a lysosomal marker in the ruffle-ended maturation ameloblasts and associated papillary cells. Morphometric analysis was carried out of the percentage area of these cells (density) occupied by the various enzyme-reactive lysosomal structures. The density of total TMPase-positive lysosomal structures, tubular lysosomes and multivesicular bodies in ruffle-ended ameloblasts were all significantly greater (p less than or equal to 0.05) in early than in late maturation enamel formation. In papillary cells the same was true of tubular lysosomes, whereas the greater density of enzyme-positive total structures in early maturation was not statistically significant when compared to late maturation. These findings demonstrate a corresponding pattern between enamel-organ lysosomal activity and the period of early enamel maturation when most enamel protein is lost. They support the likely involvement by ruffle-ended ameloblasts and papillary cells in absorption and degradation of exogenous enamel proteins.

Acid Anhydride Hydrolases↗
PubMed1990

Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV-1.

To determine whether apparently healthy persons who have had repeatedly reactive enzyme immunoassays and an indeterminate Western blot assay for antibody to the human immunodeficiency virus type 1 (HIV-1) are infected with HIV-1 or HIV-2, we studied 99 such volunteer blood donors in a low-risk area of the country. The subjects were interviewed about HIV risk factors. Coded blood specimens were tested again for HIV-1 antibody (by two different enzyme immunoassays, a Western blot assay and a radioimmunoprecipitation assay) and for HIV-2 antibody by enzyme immunoassay, for HIV-1 by the serum antigen test, for HIV-1 by culture, for human T-cell leukemia virus Type I or II antibody by enzyme immunoassay, and for sequences of HIV DNA by the polymerase chain reaction. Of the 99 blood donors, 98 reported no risk factors for HIV-1 infection; 1 donor had used intravenous drugs. After a median of 14 months (range, 1 to 30) from the time of the initial test, 65 subjects (66 percent) were still repeatedly reactive for HIV-1 antibody on at least one immunoassay. In 91 subjects (92 percent) the Western blot results were still indeterminate, whereas in 8 they were negative. No donor met the criteria for a positive Western blot assay for HIV-1, and none had evidence of HIV-1 or HIV-2 infection on culture or by any other test. We conclude that persons at low risk for HIV infection who have persistent indeterminate HIV-1 Western blots are rarely if ever infected with HIV-1 or HIV-2.

Adult↗
PubMed1983

Meldola Blue for the cytochemical demonstration of leukocyte dehydrogenases.

The new electron carrier Meldola Blue was employed in the cytochemical demonstration of succinate, lactate, and glucose-6-phosphate dehydrogenases in leukocytes. A significant increase in dehydrogenase activity, with better localization and less diffusion of the reaction product, was observed when compared to enzyme reactivity carried out without Meldola Blue. Meldola Blue can facilitate the demonstration of both bound and soluble leukocyte dehydrogenases. Current methods which could yield better visualization of leukocyte dehydrogenases, particularly succinate, lactate, and glucose-6-phosphate dehydrogenases in neutrophils, suggest excellent use can be made of Meldola Blue in the clinical laboratory. Reliable histochemical determination of these enzymes can aid in differentiating younger from older neutrophils. In addition, glucose-6-phosphate dehydrogenase might be used in conjunction with the nitroblue tetrazolium (NBT) test in separating persons with bacterial infections from those with nonbacterial illness. Furthermore, histochemical visualization of this enzyme may also prove beneficial in identifying glucose-6-phosphate dehydrogenase-deficient individuals.

Bacterial Infections↗
PubMed2006

The specific, submicromolar-Km ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn2+ as activating cation and increase in Km for ADP-ribose, both elicited by H2O2.

Free ADP-ribose is a putative second messenger and also a potentially toxic compound due to its non-enzymic reactivity towards protein side chains. ADP-ribose hydrolysis is catalysed by NDP-sugar/alcohol pyrophosphatases of differing specificity, including a highly specific, low-K(m) ADP-ribose pyrophosphatase. In humans, a submicromolar-K(m) ADP-ribose pyrophosphatase has been purified from placenta, while recombinant NUDT9 has been described as a similarly specific enzyme with a nudix motif, but with a 10(2)-10(3) higher K(m). Here, a comparative study of both proteins is presented showing that they are in fact enzymically indistinguishable; crucially, they both have submicromolar K(m) for ADP-ribose. This study firmly supports the view that the ADP-ribose pyrophosphatase present in human tissues is a product of the NUDT9 gene. In addition, this study reveals previously unknown properties of both enzyme forms. They display the same, differential properties in the presence of Mg(2+) or Mn(2+) as activating cations with respect to substrate specificity, ADP-ribose saturation kinetics, and inhibition by fluoride. Treatment with H(2)O(2) alters the Mg(2+)/Mn(2+) responses and increases the K(m) values for ADP-ribose, changes that are reversed by DTT. The results are discussed in relation to the proposed roles for ADP-ribose in oxidative/nitrosative stress and for ADP-ribose pyrophosphatase as a protective enzyme whose function is to limit the intracellular accumulation of ADP-ribose.

Adenosine Diphosphate Ribose↗
PubMed1980

Differential release of membrane-bound alkaline phosphatase isoenzymes from tumor cells by bromelain.

A rapid enzymatic method is presented which results in the selective release of cell-surface alkaline phosphatase isoenzymes. Treatment of suspensions of human tumor cell lines with the proteolytic enzyme bromelain released certain alkaline phosphatase isoenzymes into low-molecular-weight, catalytically active forms. Cells which expressed term placental or intestinal isoenzymes were equally susceptible to this treatment. A cell line which expressed early placental isoenzyme, however, was unaffected by bromelain as indicated by complete recovery of activity in the treated membrane fraction. Successful solubilization of immunologically reactive enzyme allows quantitation of levels of cell-surface enzyme in response to modulators of gene expression. Moreover, the observed selective solubilization of isoenzymes by bromelain may be of general use in analyses of the physical association between other biologically important surface proteins and the lipid components of the cell membrane.

Alkaline Phosphatase↗
PubMed1988

Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population.

Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens. The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6%. Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only [GRO]) (0.4 to 0.5% of donors). Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot. Most GRO sera reacted with p15/p17 bands on HIV immunoblot. Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E. coli-expressed p55gag. This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera. More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies.

Antibodies, Viral↗
PubMed1997

Peroxide treatment changes activity of non specific esterase in vascular endothelium of isolated pig aorta in vitro.

The effect of hydrogen peroxide on certain enzymes contained in endothelial cells was investigated in vitro. Specifically in this study the influence of hydrogen peroxide on the non-specific esterase activity in endothelial cells of the perfused pig aorta was examined. We perfused an isolated segment of the pig aorta for 10 h and added 5, 10, 12, 15 ml 10% hydrogen peroxide to 100 ml perfused medium. In unperfused pig aorta 99.34% of the endothelial cells reacted positive. After 10 h of perfusion without hydrogen peroxide administration 93.25% endothelial cells still showed positive enzyme reactivity. In presence of hydrogen peroxide in the perfusion medium the activity of non-specific esterase was progressively inhibited, finally causing complete inactivation.

Adenosine Triphosphatases↗
PubMed2006

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions.

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.

Bacillus cereus↗
PubMed1993

Specificity of Streptomyces griseus aminopeptidase and modulation of activity by divalent metal ion binding and substitution.

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass [Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471-477]. A study of the specificity of S. griseus aminopeptidase using amino acid 4-nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4-nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa-proline N-terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate-dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1,10-phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4-nitroanilide at a 10-fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4-nitroanilide at a more than 20-fold faster rate than the zinc or manganese enzymes.

Aminopeptidases↗
PubMed1994

Early detection of antibodies to human immunodeficiency virus 1 by a third-generation enzyme immunoassay. A comparative study with the results of second-generation immunoassays and western blot.

The aim of the study was to examine the sensitivity and the specificity of a HIV-1/HIV-2 third-generation enzyme immunoassay, the Abbott recombinant HIV-1/HIV-2 third-generation enzyme immunoassay, which is reported to detect simultaneously IgG and IgM. Sensitivity was evaluated with sera from seropositive subjects and a series of samples from eight HIV-1 seroconverting subjects. Results were compared with Western blot, second-generation immunoassays (including Vidas HIV-1 + 2) and an HIV-1 antigen assay. Specificity was studied with sera collected from a low-risk population and sera with false-reactive enzyme immunoassays results. In seven cases, the third-generation immunoassay detected HIV-antibodies several days earlier (range 4 to > or = 9 days) than the Western blot test according to the criteria proposed by the Association of State and Territorial Public Health Laboratory Directors, ie, positive with two of the three bands-gp160 or gp120, gp41, and p24. In the last case detection occurred at the same time as Western blot. The second-generation tests generally detected HIV-antibodies at the same time as Western blot. Antigaenemia was positive in the first samples tested in most cases, prior to or simultaneously with the Abbott third-generation test first reactivity, before the second generation tests and the Western blot test. In most cases, the disappearance of detectable HIV antigen was observed, and was concurrent with the development of the antibodies immune response. For our low-risk population, the current third-generation EIA test obtained a false-reactive rate of 0.26%. Our data indicate that the Abbott third-generation immunoassay is more sensitive than the Western blot test and the second-generation tests. The addition of a third-generation assay to the strategy for HIV-antibody screening may indeed be of interest and could make it possible to decrease the number of false-negative results.

Blotting, Western↗
PubMed1977

Nitrate reductase from Spinacea oleracea. FAD and the reactivation of the enzyme treated with p-Hydroxymercuribenzoate.

Spinach nitrate reductase complex previously inactivated by treatment with mercurials p-hydroxymercuribenzoate or p-hydroxymercuriphenyl sulphonate can be reactivated by incubation with dithioerythritol. The reactivation of NADH-diaphorase seems to be FAD-dependent, whereas that of FNH2-nitrate reductase is not. The requirement of FAD for NADH-inactivation of nitrate reductase treated with p-hydroxymercuribenzoate disappears after treatment with dithioerythritol.

Dihydrolipoamide Dehydrogenase↗
PubMed1999

Novel mechanism of inhibition of elastase by beta-lactams is defined by two inhibitor crystal complexes.

Two structurally related beta-lactams form different covalent complexes upon reaction with porcine elastase. The high resolution x-ray structures of these two complexes provide a clear insight into the mechanism of the reaction and suggest the design of a new class of serine protease inhibitors that resist enzyme reactivation by hydrolysis of the acyl intermediate. The presence of a hydroxyethyl substituent on the beta-lactam ring provides a new reaction pathway resulting in the elimination of the hydroxyethyl group and the formation of a stabilizing conjugated double bond system. In contrast, the presence of a diethyl substituent on the beta-lactam ring leads to addition of water. The two enzyme complexes show very different binding modes in the enzyme active site.

Animals↗
PubMed1979

[Mechanism of interaction between milk xanthine oxidase and p-chloromercuribenzoate. Properties of the purified enzyme].

Two types of complexes are formed during the interaction of xanthine oxidase with p-chloromercurybenzoate (pCMB). The reversible inactive complex (presumably of absorption nature) is formed practically instantaneously and competitively with regard to the substrate (Ki=6,2 . 10(-8) M) in 0,05 M phosphate buffer (pH 7,8, 25 degrees) and does not involve the fast-reacting SH-groups of the enzyme. Reactivation of xanthine oxidase is observed during prolonged incubation of the inactive complex at 0 degrees; it is associated with the interaction between pCMB and the fact-reacting SH-groups. This interaction results in a dissociation of the inactive complex. The blocking of the slow-reacting SH-groups is accompanied by an irreversible loss of the xanthine oxidase activity. The enzyme modification by blocking of 10 fast-reacting SH-groups does not involve the Fe-S clusters, but results in local changes in the enzyme conformation. This is manifested in a 2-fold increase of Km and the rate constants of proteolysis of the modified xanthine oxidase as compared to the native enzyme. The rate constants of proteolysis by trypsin for the native and modified enzymes in 0,05 M phosphate buffer (pH 7,8; 37 degrees) are 3,7 . 10(-3) min-1 and 7,0 . 10(-3) min-1, respectively; those for chymotrypsin in the same buffer (30 degrees) are 1,5 . 10(-2) min-1 and 6,0 . 10(-2) min-1, respectively.

Animals↗
PubMed1983

The reversal by oximes and their de oximinomethyl analogues of neuromuscular block produced by soman.

A series of oximes and related compounds were assessed for their ability to restore soman-induced neuromuscular block in the isolated diaphragm preparation of the rat, guinea-pig and marmoset. In the rat the bispyridinium oximes HS6, HI6 and HS14 were superior to P2S and all other compounds tested. Conversely, in the guinea-pig, most of the compounds tested produced a good reversal of neuromuscular block. In a limited number of experiments in the marmoset, only a partial reversal of neuromuscular block, was obtained with the oximes HI6 and HS6. The restoration of neuromuscular block was due to one or more of the following factors: (i) enzyme reactivation (ii) direct action (iii) adaptation. The results of this study suggest that both the acetylcholine receptor and the rate of 'ageing' of soman inhibited AChE are different in these three species.

Acetylcholinesterase↗
PubMed2001

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

Amino Acid Motifs↗
PubMed1983

HI6 as an antidote to soman poisoning in rhesus monkey respiratory muscles in-vitro.

This study was carried out to evaluate the rhesus monkey as a model for man with respect to oxime-induced acetylcholinesterase reactivation as a mechanism contributing to restoration of soman poisoned neuromuscular function. In-vitro neuromuscular blockade in intercostal and diaphragm muscle strips of rhesus monkeys was induced by exposing them for 2.5 or 15 min to the cholinesterase inhibitor soman. Subsequent treatment with the oxime HI6 produced only a partial reversal of this blockade. Only a minor part of the recovery obtained could be attributed to enzyme reactivation and suggested that this species responded in a manner closer to that reported in man than other animal species studied.

Animals↗
PubMed2001

Modulation of mitochondrial function by hydrogen peroxide.

During normal cellular metabolism, mitochondrial electron transport results in the formation of superoxide anion (O(2)) and subsequently hydrogen peroxide (H(2)O(2)). Because H(2)O(2) increases in concentration under certain physiologic and pathophysiologic conditions and can oxidatively modify cellular components, it is critical to understand the response of mitochondria to H(2)O(2). In the present study, treatment of isolated rat heart mitochondria with H(2)O(2) resulted in a decline and subsequent recovery of state 3 NADH-linked respiration. Alterations in NADH levels induced by H(2)O(2) closely paralleled changes in the rate of state 3 respiration. Assessment of electron transport chain complexes and Krebs cycle enzymes revealed that alpha-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and aconitase were susceptible to H(2)O(2) inactivation. Of particular importance, KGDH and SDH activity returned to control levels, concurrent with the recovery of state 3 respiration. Inactivation is not because of direct interaction of H(2)O(2) with KGDH and SDH. In addition, removal of H(2)O(2) alone is not sufficient for reactivation. Enzyme activity does not recover unless mitochondria remain intact. The sensitivity of KGDH and SDH to H(2)O(2)-mediated inactivation and the reversible nature of inactivation suggest a potential role for H(2)O(2) in the regulation of KGDH and SDH.

Animals↗
PubMed1991

Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence.

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

2,4-Dinitrophenol↗