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Competitive Inhibitor (competitive + inhibitor)
Kinds of Competitive Inhibitor Selected AbstractsDuplexed On-Microbead Binding Assay for Competitive Inhibitor of Epidermal Growth Factor Receptor by Quantitative Flow CytometryBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2010Wen-Jun Lan Conventional methods for evaluation of EGFR inhibitors are limited. This study describes a duplexed on-microbead binding assay allowing competitive EGFR inhibitors to be quantificationally evaluated in vitro. Polystyrene microbeads barcoded by fluoresceine isothiocyanate fluorescence as high brightness and low brightness microspheres were coated with receptor tyrosine kinase (RTK) ligand-epidermal growth factor (EGF)/stem cell factor (SCF) and ATP/GTP, respectively. High and low brightness microbeads were mixed and incubated with EGFR and its competitive inhibitor in binding assay buffer. Phycoerythrin (PE) fluorescence-labelled antibody was employed to report the level of EGFR binding to EGF/SCF and ATP/GTP. Values were numbered via PE molecules assessed by quantitative flow cytometry. Results from this study demonstrated that incubation with EGFR identified by PE-labelled antibody can make EGF- and ATP-coated microbeads luminous. And EGF or ATP-competitive EGFR inhibitors, respectively, alleviated this in a concentration-dependent manner. Coating microbeads with SCF or GTP as a negative control cannot capture EGFR. The duplexed on-microbead binding assay in this study might be useful for discovering ligand- and ATP-competitive EGFR inhibitors in a rapid and quantificational approach. [source] ChemInform Abstract: Polyhydroxylated Aziridinylcyclopentanes as Glycomimetics: A New Competitive Inhibitor of ,-Mannosidase.CHEMINFORM, Issue 2 2002Ryan C. Schoenfeld Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Nanomolar Competitive Inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor Type,II DehydroquinaseCHEMMEDCHEM, Issue 2 2007Verónica F. Abstract Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid have been synthesized and tested as inhibitors of M.,tuberculosis and S.,coelicolor type,II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross-coupling and copper(I)-catalyzed 2,3-dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para -nitrophenyl derivative is almost 20-fold more potent as a competitive inhibitor against the S.,coelicolor enzyme than that of M.,tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S.,coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4,30,nM). These derivatives are also competitive inhibitors of the M.,tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S.,coelicolor enzyme, a 6-benzothiophenyl derivative, has a Ki value of 4,nM,over 2000-fold more potent than the best previously known inhibitor, (1R,4R,5R)-1,5-dihydroxy-4-(2-nitrophenyl)cyclohex-2-en-1-carboxylic acid (8,,M), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S.,coelicolor enzyme (GOLD,3.0.1), suggest a key , -stacking interaction between the aromatic rings and Tyr,28, a residue that has been identified as essential for enzyme activity. [source] No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+ -ATPase from short-term stimulated rat muscleACTA PHYSIOLOGICA, Issue 4 2009T. Mishima Abstract Aim:, We examined whether structural alterations to the adenine nucleotide binding site (ANBS) within sarcoplasmic (endo) reticulum Ca2+ -ATPase (SERCA) would account for contraction-induced changes in the catalytic activity of the enzyme as assessed in vitro. Methods:, Repetitive contractions were induced in rat gastrocnemius by electrical nerve stimulation. Measurements of sarcoplasmic reticulum properties were performed on control and stimulated muscles immediately after or at 30 min after the cessation of 5-min stimulation. In order to examine the properties at the ANBS, the binding capacity of SERCA to fluorescence isothiocyanate (FITC), a competitive inhibitor at the ANBS, was analysed in microsomes. Results:, Short-term electrical stimulation evoked a 23.9% and 32.6% decrease (P < 0.05) in SERCA activity and in the FITC binding capacity, respectively, in the superficial region of the muscle. Whereas SERCA activity reverted to normal levels during 30-min recovery, a restoration of the FITC binding capacity did not occur. Conclusion:, The discordant changes between the enzyme activity and the FITC binding suggest that, at least during recovery after exercise, changes in SERCA activity may not correlate closely with structural alterations to the ANBS within the enzyme. [source] Biokinetics of cadmium and zinc in a marine bacterium: Influences of metal interaction and pre-exposureENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2008Dongshi Chen Abstract The uptake kinetics of Cd and Zn, as influenced by metal interaction and metal pre-exposure, was examined in the gram-positive marine bacterium Bacillus firmus over a wide range of ambient free-Cd and -Zn concentrations. Bacteria were exposed to experimental media with different concentrations of Cd and Zn over a short, 15-min period. Zinc was found to be an effective competitive inhibitor of Cd uptake when the Zn2+ concentration ([Zn2+]) was increased to 10,8 M, whereas the Cd concentration (ranging from 10,9 to 10,6 M) did not affect Zn uptake. Inhibition of Cd uptake was dependent on [Zn2+] instead of the [Zn2+] to Cd2+ concentration ratio. Cadmium uptake at different [Zn2+] was significantly inhibited by a sulfur ligand (SH) blocker (N -ethylmaleimide) and a Ca-channel blocker (lanthanum), suggesting that competition between Cd and Zn most likely occurred via binding to the same transport sites. Cadmium efflux also was determined in the presence of different [Zn2+]. A biphasic depuration of Cd was found when [Zn2+] was greater than 10,8 M, whereas the calculated Cd efflux rate was independent of [Zn2+]. We further exposed B. firmus at different Cd or Zn concentrations for 24 h, then determined the metal uptake and efflux kinetics as well as the metallothionein (MT) induction. Both the Cd and Zn cellular concentrations increased with greater exposed metal concentration, but the MT levels and efflux were little affected by the elevated metal concentration. To some extent, however, the Cd uptake was reduced with an elevated intracellular Zn concentration, suggesting that at high Cd concentrations, intracellular Zn can suppress the Cd uptake in B. firmus. These results help to understand the interactions of metals in the marine environments. [source] Competition among zinc, manganese, and cadmium uptake in the freshwater alga Scenedesmus vacuolatusENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2007Stefanie Töpperwien Abstract In the present study, Zn and Mn competition with Cd uptake was investigated in the freshwater alga Scenedesmus vacuolatus. Scenedesmus vacuolatus was exposed to experimental media with Cd and either Zn or Mn in short-term experiments; long-term experiments were undertaken to investigate the effect of growth on Cd accumulation. Cadmium accumulation in S. vacuolatus could be detected at very low exposure concentrations (free Cd2+, 2 × 10,14 to 1 × 10,11 M), and uptake was proportional to the free-Cd2+ concentration. Zinc was an effective competitive inhibitor of Cd uptake when the Zn2+ to Cd2+ ratio was greater than 14 in the exposure medium, whereas Mn competed with Cd for uptake above a Mn2+ to Cd2+ ratio of greater than 10,000. Binding constants for Cd and Zn affinity to the transport sites were determined (KZn and KCd). Values for KZn were slightly higher (log K = 9.4,9.8) than values for KCd (log K = 8.9,9.8). In contrast, Cd seemed not to compete with the Mn-binding sites for uptake over the Mn concentration range from 1 × 10,10 to 1 × 10,8 M. Determined values for the binding constants of Zn and Cd show that a simple model can be applied to predict Cd uptake at known Zn and Cd concentrations. The environmental implications of these results are discussed with respect to potential Cd toxicity for aquatic organisms. [source] Substrate specificity and inhibition of brassinin hydrolases, detoxifying enzymes from the plant pathogens Leptosphaeria maculans and Alternaria brassicicolaFEBS JOURNAL, Issue 24 2009M. Soledade C. Pedras Blackleg (Leptosphaeria maculans and Leptosphaeria biglobosa) and black spot (Alternaria brassicicola) fungi are devastating plant pathogens known to detoxify the plant defence metabolite, brassinin. The significant roles of brassinin as a crucifer phytoalexin and as a biosynthetic precursor of several other plant defences make it important in plant fitness. Brassinin detoxifying enzymes produced by L. maculans and A. brassicicola catalyse the detoxification of brassinin by hydrolysis of its dithiocarbamate group to indolyl-3-methanamine. The purification and characterization of brassinin hydrolases produced by L. maculans (BHLmL2) and A. brassicicola (BHAb) were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa. Protein characterization using LC-MS/MS and sequence alignment analyses suggested that both enzymes belong to the family of amidases with the catalytic Ser/Ser/Lys triad. Furthermore, chemical modification of BHLmL2 and BHAb with selective reagents suggested that the amino acid serine was involved in the catalytic activity of both enzymes. The overall results indicated that BHs have new substrate specificities with a new catalytic activity that can be designated as dithiocarbamate hydrolase. Investigation of the effect of various phytoalexins on the activities of BHLmL2 and BHAb indicated that cyclobrassinin was a competitive inhibitor of both enzymes. On the basis of pH dependence, sequence analyses, chemical modifications of amino acid residues and identification of headspace volatiles, a chemical mechanism for hydrolysis of the dithiocarbamate group of brassinin catalysed by BHLmL2 and BHAb is proposed. The current information should facilitate the design of specific synthetic inhibitors of these enzymes for plant treatments against blackleg and black spot fungal infections. [source] Experimental validation of metabolic pathway modelingFEBS JOURNAL, Issue 13 2008An illustration with glycolytic segments from Entamoeba histolytica In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PPi -PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PPi was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PPi was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower Vmax values for hexose-6-phosphate isomerase, PPi -PFK and aldolase were induced by PPi or ATP inhibition. It should be noted that PPi and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed ,in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties. [source] Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous estersFEBS JOURNAL, Issue 10 2008Patrick Masson The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o -nitrophenylacetanilide, o -nitrotrifluorophenylacetanilide and m -(acetamido) N,N,N -trimethylanilinium] and homologous esters (o -nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m -(acetamido) N,N,N -trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (, > , > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric ,cross-talk' between the peripheral anionic site and the catalytic centre. [source] Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin LFEBS JOURNAL, Issue 19 2003Jeffrey P. Bocock Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nm, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu. [source] Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyaseFEBS JOURNAL, Issue 14 2002Tadayoshi Kanao The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl -ACL), an ,4,4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl -ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl -ACL. His273 of the , subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA. [source] Kinetic study of sn -glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1FEBS JOURNAL, Issue 3 2002Jin-Suk Han A gene having high sequence homology (45,49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94,96 °C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H- dependent dihydroxyacetone phosphate reduction and NAD+ -dependent,glycerol-1-phosphate (Gro1P) oxidation. NADP+ -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi,bi mechanism. [source] Development and characterization of an animal model of carnitine deficiencyFEBS JOURNAL, Issue 6 2001Markus Spaniol Mammals cover their carnitine needs by diet and biosynthesis. The last step of carnitine biosynthesis is the conversion of butyrobetaine to carnitine by butyrobetaine hydroxylase. We investigated the effect of N -trimethyl-hydrazine-3-propionate (THP), a butyrobetaine analogue, on butyrobetaine hydroxylase kinetics, and carnitine biosynthesis and body homeostasis in rats fed a casein-based or a vegetarian diet. The Km of butyrobetaine hydroxylase purified from rat liver was 41 ± 9 µmol·L,1 for butyrobetaine and 37 ± 5 µmol·L,1 for THP, and THP was a competitive inhibitor of butyrobetaine hydroxylase (Ki 16 ± 2 µmol·L,1). In rats fed a vegetarian diet, renal excretion of total carnitine was increased by THP (20 mg·100 g,1·day,1 for three weeks), averaging 96 ± 36 and 5.3 ± 1.2 µmol·day,1 in THP-treated and control rats, respectively. After three weeks of treatment, the total carnitine plasma concentration (8.8 ± 2.1 versus 52.8 ± 11.4 µmol·L,1) and tissue levels were decreased in THP-treated rats (liver 0.19 ± 0.03 versus 0.59 ± 0.08 and muscle 0.24 ± 0.04 versus 1.07 ± 0.13 µmol·g,1). Carnitine biosynthesis was blocked in THP-treated rats (,0.22 ± 0.13 versus 0.57 ± 0.21 µmol·100 g,1·day,1). Similar results were obtained in rats treated with the casein-based diet. THP inhibited carnitine transport by rat renal brush-border membrane vesicles competitively (Ki 41 ± 3 µmol·L,1). Palmitate metabolism in vivo was impaired in THP-treated rats and the livers showed mixed steatosis. Steady-state mRNA levels of the carnitine transporter rat OCTN2 were increased in THP-treated rats in skeletal muscle and small intestine. In conclusion, THP inhibits butyrobetaine hydroxylase competitively, blocks carnitine biosynthesis in vivo and interacts competitively with renal carnitine reabsorption. THP-treated rats develop systemic carnitine deficiency over three weeks and can therefore serve as an animal model for human carnitine deficiency. [source] Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporusFEBS JOURNAL, Issue 16 2000Neelam A. Desai An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 °C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA , 3,AMP , RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5,dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5,dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5,dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3, or the 5, end, indicates that C-linkages are resistant to cleavage by nuclease Bh1. [source] Effects of cadmium on manganese peroxidaseFEBS JOURNAL, Issue 6 2000Competitive inhibition of MnII oxidation, thermal stabilization of the enzyme Inhibition of manganese peroxidase by cadmium was studied under steady-state and transient-state kinetic conditions. CdII is a reversible competitive inhibitor of MnII in the steady state with Ki , 10 µm. CdII also inhibits enzyme-generated MnIII,chelate-mediated oxidation of 2,6-dimethoxyphenol with Ki , 4 µm. CdII does not inhibit direct oxidation of phenols such as 2,6-dimethoxyphenol or guaiacol (2-methoxyphenol) in the absence of MnII. CdII alters the heme Soret on binding manganese peroxidase and exhibits a Kd , 8 µm, similar to Mn (Kd , 10 µm). Under transient-state conditions, CdII inhibits reduction of compound I and compound II by MnII at pH 4.5. However, CdII does not inhibit formation of compound I nor does it inhibit reduction of the enzyme intermediates by phenols in the absence of MnII. Kinetic analysis suggests that CdII binds at the MnII -binding site, preventing oxidation of MnII, but does not impair oxidation of substrates, such as phenols, which do not bind at the MnII -binding site. Finally, at pH 4.5 and 55 °C, MnII and CdII both protect manganese peroxidase from thermal denaturation more efficiently than CaII, extending the half-life of the enzyme by more than twofold. Furthermore, the combination of half MnII and half CdII nearly quadruples the enzyme half-life over either metal alone or either metal in combination with CaII. [source] S-adenosylhomocysteine sensitizes to TNF-, hepatotoxicity in mice and liver cells: A possible etiological factor in alcoholic liver diseaseHEPATOLOGY, Issue 4 2004Zhenyuan Song In alcoholic liver disease, tumor necrosis factor-, (TNF,) is a critical effector molecule, and abnormal methionine metabolism is a fundamental acquired metabolic abnormality. Although hepatocytes are resistant to TNF,-induced killing under normal circumstances, previous studies have shown that primary hepatocytes from rats chronically fed alcohol have increased TNF, cytotoxicity. Therefore, there must be mechanisms by which chronic alcohol exposure "sensitizes" to TNF, hepatotoxicity. S-adenosylhomocysteine (SAH) is product of methionine in transsulfuration pathway and a potent competitive inhibitor of most methyltransferases. In this study, we investigated the effects of increased SAH levels on TNF, hepatotoxicity. Our results demonstrated that chronic alcohol consumption in mice not only decreased hepatic S-adenosylmethionine levels but also increased hepatic SAH levels, which resulted in a significantly decreased S-adenosylmethionine-to-SAH ratio. This was associated with significant increases in hepatic TNF, levels, caspase-8 activity, and cell death. In vitro studies demonstrated that SAH-enhancing agents sensitized hepatocytes to TNF, killing, and the death was associated with increased caspase-8 activity, which was blocked by a caspase-8 inhibitor. In addition, increased intracellular SAH levels had no effect on nuclear factor ,B activity induced by TNF,. In conclusion, these results provide a new link between abnormal methionine metabolism and abnormal TNF, metabolism in alcoholic liver disease. Increased SAH is a potent and clinically relevant sensitizer to TNF, hepatotoxicity. These data further support improving the S-adenosylmethionine-to-SAH ratio and removal of intracellular SAH as potential therapeutic options in alcoholic liver disease. Supplementary material for this article can be found on the HEPATOLOGYwebsite (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;40:989,997.) [source] Tyrosine phosphorylation of the GluR2 subunit is required for long-term depression of synaptic efficacy in young animals in vivoHIPPOCAMPUS, Issue 8 2007Christopher J. Fox Abstract The study of the intracellular mechanics that underlay changes in synaptic efficacy is a rapidly evolving field of research. It is currently believed that NMDA receptors play a significant role in the induction of synaptic plasticity, whereas AMPA receptors play a significant role in its expression. For AMPA receptors, it has been shown that tyrosine phosphorylation of the GluR2 carboxyl termini is required for the expression of long-term depression of synaptic efficacy (LTD) in vitro (Ahmadian et al. (2004) EMBO J 23:1040,1050). In the present study, we sought to determine whether similar mechanisms are involved in vivo, where different stimulation parameters are required for the induction of LTD. We initially used a paired-burst (PB) paradigm that reliably induces LTD in vivo. In these animals we were able to prevent the induction and expression of PB-LTD by administering a peptide (GluR-3Y) that acted as a competitive inhibitor of tyrosine phosphorylation. In a separate set of animals, we exposed animals to brief periods of stress (S) before using low-frequency stimuli to induce LTD (S-LTD). Again, GluR2,3Y blocked both the induction and expression of S-LTD. In contrast, an inert version of the peptide, with alanine replacing the three tyrosine residues, did not inhibit LTD induction. In addition, we demonstrated that GluR2,3Y did not affect the induction of long-term potentiation in vivo. These findings support the hypothesis that tyrosine phosphorylation and AMPA receptor endocytosis are necessary steps for the induction and maintenance of two forms of LTD in the CA1 region. © 2007 Wiley-Liss, Inc. [source] Inhibitory activity of brown algal phlorotannins against hyaluronidaseINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2002Toshiyuki Shibata The inhibitory effect of brown algal phlorotannins on hyaluronidase was evaluated by an in vitro assay. Crude phlorotannins from the brown algae Eisenia bicyclis and Ecklonia kurome had a stronger inhibitory effect than well-known inhibitors such as catechins and sodium cromoglycate. IC50 values of the following six phlorotannins: phloroglucinol, an unknown tetramer, eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8,-bieckol (hexamers), were 280, 650, >800, 140, 120 and 40 ,M, respectively. The IC50 of catechin, epigallocatechin gallate and sodium cromoglycate was 620, 190 and 270 ,M, respectively. 8,8,-Bieckol, the strongest HAase inhibitor in this study, acted as a competitive inhibitor with an inhibition constant (Ki) of 35 ,M. Acetylation of the phlorotannins markedly decreased their inhibitory potency. [source] On the binding mode of urease active site inhibitors: A density functional studyINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 11 2008M. Leopoldini Abstract The way with which boric acid, a rapid reversible competitive inhibitor, binds the urease active site was explored at density functional B3LYP level of theory. The catalytic core of the enzyme was simulated by two models of different size. In both cases, amino acid residues belonging to the inner and to the outer coordination spheres of nickel ions were replaced by smaller molecular species. Contrary to the experimental indication that attributes the inhibitory ability of this acid to the lack of a nucleophilic attack by the enzyme to the boron atom, we instead found that another possibility exists based on the presence of a strong covalent , bond between boron and urease that we think can be hardly broken to allow any course of the reaction. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008 [source] Nitric Oxide: The "Second Messenger" of InsulinIUBMB LIFE, Issue 5 2000Nighat N. Kahn Abstract Incubation of various tissues, including heart, liver, kidney, muscle, and intestine from mice and erythrocytes or their membrane fractions from humans, with physiologic concentration of insulin resulted in the activation of a membrane-bound nitric oxide synthase (NOS). Activation of NOS and synthesis of NO were stimulated by the binding of insulin to specific receptors on the cell surface. A Lineweaver-Burk plot of the enzymatic activity demonstrated that the stimulation of NOS by insulin was related to the decrease in the Km for L-arginine, the substrate for NOS, with a simultaneous increase of Vmax. Addition of NG-nitro-L-arginine methyl ester (LNAME), a competitive inhibitor of NOS, to the reaction mixture completely inhibited the hormone-stimulated NO synthesis in all tissues. Furthermore, NO had an insulin-like effect in stimulating glucose transport and glucose oxidation in muscle, a major site for insulin action. Addition of NAME to the reaction mixture completely blocked the stimulatory effect of insulin by inhibiting both NO production and glucose metabolism, without affecting the hormone-stimulated tyrosine or phosphatidylinositol 3-kinases of the membrane preparation. Injection of NO in alloxan-induced diabetic mice mimicked the effect of insulin in the control of hyperglycemia (i.e., lowered the glucose content in plasma). However, injection of NAME before the administration of insulin to diabetic-induced and nondiabetic mice inhibited not only the insulin-stimulated increase of NO in plasma but also the glucose-lowering effect of insulin. [source] Kojic acid reduces the cytotoxic effects of sulfur mustard on cultures containing human melanoma cells in vitroJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2001C. N. Smith Abstract In vivo experiments have shown that melanocytes are more sensitive than keratinocytes to the cytotoxic effects of sulfur mustard when it is applied topically to pig skin.1 It has been hypothesized that this is caused by the uncoupling of the melanogenic pathway by depletion of cellular glutathione, resulting in the uncontrolled production of cytotoxic quinone free-radical species by tyrosinase.2. In the present study, the feasibility of blocking the melanogenic pathway as a means of reducing the cytotoxicity of sulfur mustard was evaluated using kojic acid. Kojic acid is a topically applied depigmenting agent that exerts its effect by acting as a slow-binding, competitive inhibitor of tyrosinase.3 Preincubation of G361 pigmented melanoma cells and mixed cultures of G361 cells and SVK keratinocytes with 2.5 mM kojic acid resulted in significant increases in the viability of these cultures as determined by neutral red (NR) and gentian violet (GV) dye binding assays for up to 48 h following exposure to 50 µM sulfur mustard. The highest levels of protection were seen in the G361 cultures, with a 26.8% increase in culture viability (NR assay) compared with the sulfur-mustard-only controls at 24 h. Preincubation of SVK cells alone with kojic acid resulted in lower increases in viability (2.5% at 24 h by the NR assay). Inhibition of the melanogenic pathway reduces the sensitivity of cultures containing pigment cells to sulfur mustard. © Crown copyright 2001. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd. [source] Phosphate and calcium are required for TGF,-mediated stimulation of ANK expression and function during chondrogenesisJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Paulina Oca The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGF,. The purpose of this study was to determine whether TGF, stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGF, increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na+/Pi channels Pit-1 and Pit-2, indicated that the stimulation of ANK expression by TGF, required the influx of phosphate, specifically by the Pit-1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGF, on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGF,. Since previous studies of endochondral ossification in the growth plate have shown that L-type calcium channels are essential for chondrogenesis, we investigated their role in the TGF,-stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L-type channel Cav1.2 (,1C) inhibited the TGF, stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGF, stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation. J. Cell. Physiol. 224: 540,548, 2010. © 2010 Wiley-Liss, Inc. [source] PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate,matrix interactionsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2001John H. Chon PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P,<,0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P,<,0.01) and heparan sulfate (HS) (P,<,0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P,<,0.05) and a reduction in cell speed (P,<,0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Futhermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes. © 2001 Wiley-Liss, Inc. [source] Sequence flexibility of the immunodominant HLA A*0201 restricted ppUL83 CD8 T-cell epitope of human cytomegalovirusJOURNAL OF MEDICAL VIROLOGY, Issue 1 2010Jakub Kopycinski Abstract The cytomegalovirus ppUL83 protein contains an immunodominant A*0201 restricted epitope between residues 495 and 503. We investigated the tolerance of this epitope to sequence variation in the context of peptide binding to HLA A*0201 and the ability to induce an Interferon gamma (IFN,) response through engagement with the T-cell receptor (TCR). The majority of mutations investigated resulted in a decrease in the production of IFN, indicating that if such variants occurred in vivo they would not be recognized by CD8 T-cell clones specific for the wild-type epitope. The mechanistic basis for the majority of the mutant peptides was their failure to bind and stabilize class I HLA cell surface expression. However, one peptide with a mutation at the P5 position (methionine to cysteine) resulted in a significant enhanced binding to HLA A*0201 and also an increase in cell surface expression over the wild-type peptide but was unable to engage with the CD8 TCR and trigger IFN, production. This peptide acted as a competitive inhibitor of the wild-type peptide but could not fully inhibit IFN, production by the latter. We subsequently investigated whether mutations of the HLA A*0201 epitope were evident in immunocompromized patients experiencing either rapid exponential or persistent cytomegalovirus replication. J. Med. Virol. 82:94,103, 2010. © 2009 Wiley-Liss, Inc. [source] Modeling an active conformation for linear peptides and design of a competitive inhibitor for HMG-CoA reductaseJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2008Valeriy V. Pak Abstract This study presents an approach that can be used to search for lead peptide candidates, including unconstrained structures in a recognized sequence. This approach was performed using the design of a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In a previous design for constrained peptides, a head-to-tail cyclic structure of peptide was used as a model of linear analog in searches for lead peptides with a structure close to an active conformation. Analysis of the conformational space occupied by the peptides suggests that an analogical approach can be applied for finding a lead peptide with an unconstrained structure in a recognized sequence via modeling a cycle using fixed residues of the peptide backbone. Using the space obtained by an analysis of the bioactive conformations of statins, eight cyclic peptides were selected for a peptide library based on the YVAE sequence as a recognized motif. For each cycle, the four models were assessed according to the design criterion ("V" parameter) applied for constrained peptides. Three cyclic peptides (FGYVAE, FPYVAE, and FFYVAE) were selected as lead cycles from the library. The linear FGYVAE peptide (IC50,=,0.4,µM) showed a 1200-fold increase the inhibitory activity compared to the first isolated LPYP peptide (IC50,=,484,µM) from soybean. Experimental analysis of the modeled peptide structures confirms the appropriateness of the proposed approach for the modeling of active conformations of peptides. Copyright © 2008 John Wiley & Sons, Ltd. [source] Structure,Activity Relationships Among N -Arachidonylethanolamine (Anandamide) Head Group Analogues for the Anandamide TransporterJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Abbas Jarrahian Abstract: Two putative endocannabinoids, N -arachidonylethanolamine (AEA) and 2-arachidonylglycerol, are inactivated by removal from the extracellular environment by a process that has the features of protein-mediated facilitated diffusion. We have synthesized and studied 22 N-linked analogues of arachidonylamide for the purpose of increasing our understanding of the structural requirements for the binding of ligands to the AEA transporter. We have also determined the affinities of these analogues for both the CB1 cannabinoid receptor and fatty acid amide hydrolase (FAAH). We have identified several structural features that enhance binding to the AEA transporter in cerebellar granule cells. We have confirmed the findings of others that replacing the ethanolamine head group with 4-hydroxybenzyl results in a high-affinity ligand for the transporter. However, we find that the same molecule is also a competitive inhibitor of FAAH. Similarly, replacement of the ethanolamine of AEA with 3-pyridinyl also results in a high-affinity inhibitor of both the transporter and FAAH. We conclude that the structural requirements for ligand binding to the CB1 receptor and binding to the transporter are very different; however, the transporter and FAAH share most, but not all, structural requirements. [source] Tumor necrosis factor-, mediates polymethylmethacrylate particle-induced NF-,B activation in osteoclast precursor cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2002John C. Clohisy Tumor necrosis factor-, (TNF) is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of implant particle-induced osteolysis. The nuclear transcription factor NF-,B mediates TNF signaling and this transcription complex is necessary for osteoclastogenesis. Because polymethylmethacrylate (PMMA) particles cause osteolysis, we reasoned the PMMA would induce NF-,B activation. In fact, we find that exposure of osteoclast precursors, in the form of colony stimulating factor-1 (CSF-1) dependent murine bone marrow macrophages, to PMMA particles prompts nuclear translocation and activation of NF-,B. Supershift assays confirm the presence of the p50 and p65 NF-,B subunits in the activated transcription factor. Particle-induced NF-,B activation is equal in both wild type and LPS- hyporesponsive cells indicating that the phenomenon does not represent endotoxin contamination. A soluble, competitive inhibitor of TNF (huTNF:Fc) dampens particle-directed NF-,B activation and this response is also abrogated in TNF,/, osteoclast precursors. Thus, PMMA particle activation of NF-,B is a secondary event resulting from enhanced TNF expression and is independent of LPS contamination. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Conformation of N-terminal HIV-1 tat (fragment 1,9) peptide by NMR and MD simulationsJOURNAL OF PEPTIDE SCIENCE, Issue 11 2001Meena Kanyalkar Abstract The N -terminal portion of HIV-1 Tat covering residues 1,9 is a competitive inhibitor of dipeptidyl peptidase IV (DP IV). We have used 1H NMR techniques, coupled with molecular dynamics methods, to determine the conformation of this peptide in the three diverse media: DMSO-d6, water (pH 2.7) and 40% HFA solution. The results indicate that in both DMSO-d6 and HFA the peptide has a tendency to acquire a type I ,-turn around the segment Asp5 -Pro6 -Asn7 -Ile8. The N -terminal end is seen to be as a random coil. In water, the structure is best described as a left-handed polyproline type II (PPII) helix for the mid segment region Asp2 to Pro6. The structures obtained in this study have been compared with an earlier report on Tat (1,9). Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd. [source] Key Role of Ethanol-Derived Acetaldehyde in the Motivational Properties Induced by Intragastric Ethanol: A Conditioned Place Preference Study in the RatALCOHOLISM, Issue 2 2008Alessandra T. Peana Background:, Acetaldehyde (ACD), the first metabolite of ethanol (EtOH), is produced peripherally by gastric and hepatic alcohol dehydrogenase (ADH) and centrally by brain catalase. In spite of the aversive properties classically ascribed to ACD, it has recently been suggested that ACD might mediate some of the motivational effects of EtOH. Accordingly, the relative role of ACD in the positive motivational properties of EtOH ingested is increasingly becoming the matter of debate. Thus, we studied the ability of intragastrically administered EtOH, ACD and EtOH-derived ACD to induce conditioned place preference (cpp) in rats. Methods:, Wistar rats were pretreated intraperitoneally with saline, the peripheral competitive inhibitor of ADH, 4-methylpyrazole (4-MP, 22.5, 45 or 67.5 mg/kg) or with the selective ACD-sequestrating agent, d -penicillamine (DP, 25 or 50 mg/kg), before the intragastric administration of saline, EtOH (0.5, 1 or 2 g/kg) or ACD (10, 20, or 40 mg/kg). The specificity of 4-MP and DP effects was addressed using morphine-induced cpp (2.5 mg/kg). Results:, Both, EtOH and ACD dose-dependently induced cpp; further, while EtOH-induced cpp was prevented by the administration of 4-MP and by DP, ACD-induced cpp was unaltered by 4-MP administration and prevented by DP. Both pretreatments did not interfere with morphine-induced cpp indicating that 4-MP and DP specifically modulate the motivational properties of EtOH and ACD. Conclusion:, The ability of 4-MP and DP to decrease EtOH-induced cpp suggests that a reduction of ACD levels is crucial in depriving EtOH from its motivational properties as indexed by the cpp procedure. In addition, this conclusion is supported by the inefficacy of 4-MP in preventing ACD-induced cpp, and by its blockade observed after administration of the selective ACD sequestrating agent DP. The present results underscore the role of EtOH-derived ACD in EtOH-induced motivational properties as well as its abuse liability. [source] Modelling the effect of superatmospheric oxygen concentrations on in vitro mushroom PPO activity,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2006Perla A Gómez Abstract The kinetics of polyphenol oxidase (PPO, EC 1.14.18.1) with respect to oxygen concentrations from 5 to 100% using chlorogenic acid (CGA) as substrate was examined. In vitro mushroom PPO activity was determined by measuring the consumption of oxygen during the oxidation reaction. A differential Michaelis,Menten model was fitted to the obtained total depletion curves. The product concentration as well as the concentration of oxygen had a clear inhibitory effect on the reaction rate. However, the inhibitory effect of oxygen was more evident at low product concentration. A linear mixed inhibition model that considered both the product (oxidised CGA) and oxygen as inhibitors was developed. A model with the product as a competitive inhibitor and oxygen as an uncompetitive inhibitor was the most appropriate to explain the reaction kinetics. The values of the inhibition constants calculated from the model were 0.0032 mmol L,1 for Km (Michaelis,Menten constant related to oxygen), 0.023 mmol L,1 for Kmc (constant for competitive inhibition due to the product), 1.630 mmol L,1 for Kmu (constant for uncompetitive inhibition due to oxygen) and 1.77 × 10,4 mmol L,1 s,1 for Vmax (maximum reaction rate). The results indicate that superatmospheric oxygen concentrations could be effective in preventing enzymatic browning by PPO. Copyright © 2006 Society of Chemical Industry [source] | |||||||||||||||||||||||||||||||