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Results for “Enzyme Reactivators”

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PubMed1984

Hydroxyproline 2-epimerase of Pseudomonas. Subunit structure and active site studies.

Hydroxyproline 2-epimerase of Pseudomonas putida was purified to homogeneity by an improved procedure. The native enzyme consists of two probably identical subunits. Alkylation of the active site with labeled reagents resulted in the loss of 80-85% of the activity but the incorporation of only one alkyl group even though the active site contains a Cys residue from each of the two subunits. This result suggests that the enzyme shows half-site reactivity. The labeled enzyme was further subjected to exhaustive alkylation with unlabeled iodoacetate, permitting tryptic hydrolysis and isolation of an active site peptide in 30% yield. The specific radioactivity of the peptide was consistent with the first result, that only 1 mol of alkyl group was initially incorporated into active site. The active site peptide (14 residues) was sequenced and found to possess homology with the clostridial proline racemase.

Alkylation↗

PubMed1979

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.

Animals↗

PubMed2000

Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b.

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.

Animals↗

PubMed1982

A rapid and sensitive solid-phase enzyme immunoassay for C-reactive protein.

C-reactive protein (CRP) is an acute-phase reactant the concentration of which increases significantly following tissue injury or inflammation. I report here the development of a rapid and sensitive solid-phase enzyme immunoassay (EIA) for determination of serum CRP. The inclusion of 4% (w/v) polyethylene glycol (PEG) in the diluent buffers made it possible to quantitate CRP within 4 h at room temperature. The minimum detectable amount was about 500 pg CRP/ml. Excellent correlation in determination of serum CRP was found between solid-phase EIA and nephelometric assay, radial immunodiffusion and the qualitative latex agglutination tests.

Animals↗

PubMed2003

A novel mechanism for activation of the protein kinase Aurora A.

Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly [14-16]. Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly.

Animals↗

PubMed1970

Purification and properties of alpha-D-mannosidase from the limpet, Patella vulgata.

1. alpha-Mannosidase from the limpet, Patella vulgata, was purified nearly 150-fold, with 40% recovery. beta-N-Acetylglucosaminidase was removed from the preparation by treatment with ethanol. The final product was virtually free from beta-galactosidase. 2. Limpet alpha-mannosidase was assayed at pH3.5 and at this pH it was necessary to add Zn(2+) for full activity. At pH5, the enzyme had the same activity in the presence or absence of added Zn(2+). 3. On incubation at acid pH, the enzyme underwent reversible inactivation, which was prevented by adding Zn(2+). 4. EDTA accelerated inactivation and the addition of Zn(2+) at once restored activity. No other cation was found to reactivate the enzyme. 5. Cl(-) had an unspecific effect on hydrolysis by limpet alpha-mannosidase. It increased the rate of reaction with substrate. The anion did not prevent or reverse inactivation by EDTA. 6. It is concluded that alpha-mannosidase is a metalloenzyme or enzyme-metal ion complex, dissociable at the pH of activity, and that it requires Zn(2+) specifically.

Animals↗

PubMed1991

Activation of the matrix metalloproteinase inhibitor complex by a low molecular weight angiogenic factor.

Tissue inhibitor of matrix metalloproteinases is the major inhibitor of the collagenolytic enzymes and the inhibitory complex has been thought to be irreversible. In this paper we show that a low molecular weight non-protein endothelial cell stimulating angiogenic factor is able to reactivate the enzyme from the inhibitor complex and liberate free inhibitor. The importance of an angiogenic factor able to initiate limited degradation of extra-cellular matrix such that space is created for new capillary growth is discussed.

Angiogenesis Inducing Agents↗

PubMed2005

Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells.

Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells.

Arachidonate 5-Lipoxygenase↗

PubMed1998

Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase celB.

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 A resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid-base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032-16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters kinact=0.29+/-0.02 min-1 and Kinact=0.72+/-0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (Ki=9+/-1 mM), a behaviour that was also observed with the substrate 2',4'-dinitrophenyl beta-D-cellobioside (kcat=40+/-1 s-1, Km=0.35+/-0.03 mM, Ki=24+/-4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (kH2O=0.0022 min-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (kreact=0.024 min-1, Kreact=54 mM). Digestion of the labelled enzyme by pepsin followed by LC-ESI-tandem MS (MS-MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation of this peptide by HPLC and subsequent collision-induced fragmentation by ESI-MS-MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid-base catalyst Glu-203 are conserved in all known family-12 sequences.

Amino Acid Sequence↗

PubMed1980

Glutamate dehydrogenase from peas: isolation, quaternary structure, and influence of cations on activity.

The most active multiple molecular form of glutamate dehydrogenase from pea seeds has been enriched 30000-fold with a recovery of 60--70%. The preparation is homogenous as judged from gel electrophoresis of native and dodecylsulfate-denatured enzyme and from analytical ultracentrifugation. Specific activities were 530 U/mg in the reductive amination and 90 U/mg in the oxidative deamination reaction. A sedimentation coefficient of s20, w = 10.49 S was determined. The specific volume and the molecular weight of the native enzyme were found to be v2 = 0.759 cm3/g and Mr = 260000, respectively, by equilibrium sedimentation in H2O and 90% 2H2O buffers. Dodecylsulfate electrophoresis of the denatured enzyme yielded a molecular weight of Mr = 44000 for the polypeptide chain. From our data we propose glutamate dehydrogenase from peas to be a hexamer. The hexameric structure is confirmed by the appearance of six electrophoretic bands after cross-linking with diimidates. Enzyme which was treated with Dowex A1 chelating resin was found to be almost completely inactive in the absence of divalent metal ions. From several metals tested, calcium was most efficient in reactivating the enzyme; half-maximal activity was attained at about 5 microM calcium. In contrast to potassium ions, sodium ions were found to interfere with this regulatory mechanism by activating the enzyme at high concentrations.

Animals↗

PubMed1985

Monoclonal antibodies to rabbit brain acetylcholinesterase: selective enzyme inhibition, differential affinity for enzyme forms, and cross-reactivity with other mammalian cholinesterases.

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.

Acetylcholinesterase↗

PubMed1991

Purification and characterization of peroxisomal L-pipecolic acid oxidase from monkey liver.

L-Pipecolic acid oxidase has been purified to near homogeneity from Rhesus monkey liver. The protein, a yellow monomer, has a molecular weight of 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.9. It contains a covalently bound flavin with absorption maxima at 457 and 383 nm and a shoulder at 480 nm. The purified enzyme is most reactive toward L-pipecolic acid, with lesser reactivities toward L-proline and sarcosine. The enzyme has no significant reactivity toward the D-enantiomer of pipecolic acid or toward any other amino acid tested. Benzoic acid is a competitive inhibitor of the enzyme with a Ki of 750 microM. The Km of the purified enzyme is 3.7 mM for L-pipecolic acid. With less purified preparations, the reaction product is alpha-aminodipic acid. The purified enzyme, however, produces an intermediate which reacts with ortho-aminobenzaldehyde to form an alpha-aminoadipic acid semialdehyde adduct. Thus, the formation of alpha-aminoadipic acid requires at least two enzymes.

Animals↗

PubMed1985

Phosphorylation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and modulation of its enzymic activity by calcium-activated and phospholipid-dependent protein kinase.

A calcium-activated and phospholipid-dependent protein kinase (protein kinase C) catalyzes the phosphorylation of both insoluble microsomal (Mr approximately 100,000) and purified soluble (Mr = 53,000) 3-hydroxy-3-methylglutaryl coenzyme A reductase. The phosphorylation and concomitant inactivation of enzymic activity of HMG-CoA reductase was absolutely dependent on Ca2+, phosphatidylserine, and diolein. Dephosphorylation of phosphorylated HMG-CoA reductase was associated with the loss of protein bound radioactivity and reactivation of enzymic activity. Maximal phosphorylation of purified HMG-CoA reductase was associated with the incorporation of 1.05 +/- 0.016 mol of phosphate/mol of native form of HMG-CoA reductase (Mr approximately 100,000). The apparent Km for purified HMG-CoA reductase and histone H1 was 0.08 mg/ml, and 0.12 mg/ml, respectively. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate stimulated the protein kinase C-catalyzed phosphorylation of HMG-CoA reductase. Increased phosphorylation of HMG-CoA reductase by phorbol 12-myristate 13-acetate suggests a possible in vivo protein kinase C-mediated mechanism for the short-term regulation of HMG-CoA reductase activity. The identification of the protein kinase C system in addition to the reductase kinase-reductase kinase kinase bicyclic cascade systems for the modulation of the enzymic activity of HMG-CoA reductase may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.

Adenosine Triphosphate↗

PubMedDate not supplied

A Genetically engineered cell-based system for detecting metabolism-mediated toxicity.

Xenobiotics undergoing bioactivation by CYP450 enzymes form reactive metabolites that may exert direct metabolism-mediated toxicity. An in vitro model was developed to study the direct toxic effects that follow the metabolic activation of chemicals. The model uses monolayer cultures of genetically engineered NIH-3T3 or V79 cells that express individual human or rat CYP450 isoforms, respectively. Following exposure to 1,3-dichloropropanol or cyclophosphamide, basal cytotoxicity endpoints, including neutral red uptake and Alamar Blue( reduction were used to assess changes in cell number and functional viability resulting from the formation of metabolites. Cell lines that express cytochrome P450 enzymes metabolised the test compounds, leading to increased toxicity compared with that observed in the control cell line. The use of specific inhibitors confirmed that the formation of reactive metabolites was CYP450-isoform dependent. These results indicate that a panel of genetically engineered cell lines expressing various cytochrome P450 enzyme isoforms can be used to reveal measurable metabolising capabilities, and could become a useful tool for the detection and possible determination of CYP450 isoforms in human liver metabolism-mediated toxicity.

3T3 Cells↗

PubMed1992

Isolated cells in the study of the molecular mechanisms of reperfusion injury.

Isolated cells make it possible to study mechanisms of cell and tissue injury under well-defined conditions, including the interaction of different cells in coculture experiments. Isolated cells, either in suspension or in monolayer cultures, have also been used to study the mechanism of reperfusion injury--in this case better termed as reoxygenation injury in view of the experimental approach taken. In hepatocytes, Kupffer, and endothelial cells, reoxygenation injury resulted in necrosis primarily mediated by reactive oxygen species released by various sources such as mitochondria (hepatocytes) and NADPH oxidase (Kupffer cells). In contrast, contracture was a characteristic feature of reoxygenation injury occurring in cardiomyocytes without loss of cytosolic enzymes. Beside reactive oxygen species, Kupffer cells were activated to release prostanoids and a decrease in endothelial cell-mediated fibrinolysis occurred upon reoxygenation. Reoxygenation injury in endothelial cells was significantly increased when neutrophils were added at the time of reoxygenation, presumably due to additional generation of reactive oxygen species and the release of proteases. As exemplified for the liver, these experiments suggest a mechanism of reperfusion injury in which the various cell types of a given tissue differ significantly in their response to hypoxia-reoxygenation but in which they interact with each other in a complex pathobiochemical network via various mediators such as cytokines, and tissue damaging effector molecules such as reactive oxygen species. Future experiments with isolated cells will allow detailed analysis of the underlying molecular mechanisms.

Cells, Cultured↗

PubMed1996

A cellular automata model of enzyme kinetics.

We have developed a cellular automata model of an enzyme reaction with a substrate in water. The model produces Michaelis-Menten kinetics with good Lineweaver-Burk plots. The variation in affinity parameters predicts that, in general, hydrophobic substrates are more reactive with enzymes, this attribute being more important than the relationship between enzyme and substrate. The ease of generation and the illustrative value of the model lead us to believe that cellular automata models have a useful role in the study of dynamic phenomena such as enzyme kinetics.

Automation↗

PubMed1995

Role of arginine residues of bovine liver dihydrodiol dehydrogenase 2 in the binding of anionic substrates.

Bovine liver dihydrodiol dehydrogenase (DD2) was inactivated following pseudo-first order manner by the treatment of 5 mM 2,3-butanedione (BD) as functions of incubation-time and concentration. Anionic substrates or analog which have a carboxyl group, D-glucuronate, p-carboxybenzaldehyde and D-glycerate, protected DD2 efficiently. But, the other substrates or coenzymes and their analogs did not show any protection on the inactivation, i.e., D,L-glyceraldehyde, D-erythrose, NADP+, NAD+, 2',5'-ADP, 2'-AMP. Results of kinetic analyses suggest that the inactivated enzyme lost its binding ability to anionic substrates. The inactivated enzyme was reactivated very effectively by removing excess BD by gelfiltration for 30 min.

Animals↗

PubMed2002

GroEL interacts transiently with oxidatively inactivated rhodanese facilitating its reactivation.

When the enzyme rhodanese was inactivated with hydrogen peroxide (H(2)O(2)), it underwent significant conformational changes, leading to an increased exposure of hydrophobic surfaces. Thus, this protein seemed to be an ideal substrate for GroEL, since GroEL uses hydrophobic interactions to bind to its substrate polypeptides. Here, we report on the facilitated reactivation (86%) of H(2)O(2)-inactivated rhodanese by GroEL alone. Reactivation by GroEL required a reductant and the enzyme substrate, but not GroES or ATP. Further, we found that GroEL interacted weakly and/or transiently with H(2)O(2)-inactivated rhodanese. A strong interaction with rhodanese was obtained when the enzyme was pre-incubated with urea, indicating that exposure of hydrophobic surfaces alone on oxidized rhodanese was not sufficient for the formation of a strong complex and that a more unfolded structure of rhodanese was required to interact strongly with GroEL. Unlike prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that GroEL can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein. Additionally, the mechanism for the GroEL-facilitated reactivation of rhodanese shown here appears to be different than that for the chaperonin-assisted folding of chemically unfolded polypeptides in which a nucleotide and sometimes GroES is required.

Adenosine Triphosphate↗