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PubMed2006

Antibodies against beta-amyloid reduce Abeta oligomers, glycogen synthase kinase-3beta activation and tau phosphorylation in vivo and in vitro.

Although active and passive immunization against the beta-amyloid peptide (Abeta) of amyloid plaque-bearing transgenic mice markedly reduces amyloid plaque deposition and improves cognition, the mechanisms of neuroprotection and impact on toxic oligomer species are not understood. We demonstrate that compared to control IgG2b, passive immunization with intracerebroventricular (icv) anti-Abeta (1-15) antibody into the AD HuAPPsw (Tg2576) transgenic mouse model reduced specific oligomeric forms of Abeta, including the dodecamers that correlate with cognitive decline. Interestingly, the reduction of soluble Abeta oligomers, but not insoluble Abeta, significantly correlated with reduced tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta), a major tau kinase implicated previously in mediating Abeta toxicity. A conformationally-directed antibody against amyloid oligomers (larger than tetramer) also reduced Abeta oligomer-induced activation of GSK3beta and protected human neuronal SH-SY5Y cells from Abeta oligomer-induced neurotoxicity, supporting a role for Abeta oligomers in human tau kinase activation. These data suggest that antibodies that are highly specific for toxic oligomer subspecies may reduce toxicity via reduction of GSK-3beta, which could be an important strategy for Alzheimer's disease (AD) therapeutics.

Amyloid beta-Peptides↗

PubMed1998

Essential histidyl residues at the active site(s) of sucrose-phosphate synthase from Prosopis juliflora.

Chemical modification of sucrose-phosphate synthase (EC 2.4.1.14) from Prosopis juliflora by diethyl pyrocarbonate (DEP) and photo-oxidation in the presence of rose bengal (RB) which modify the histidyl residues of the protein resulted in the inactivation of the enzyme activity. This inactivation was dependent on the concentration of the modifying reagent and the time of incubation and followed pseudo-first order kinetics. For both the reagents, the inactivation was maximum at pH 7.5, which is consistent with the involvement and presence of histidine residues at the active site of the enzyme. Substrates, UDPG and F6P protected the enzyme against the inactivation by the modifying reagents suggesting that the histidine residues may be involved in the binding of these substrates and are essential for the catalytic activity. Specificity of DEP was indicated by an increase in absorbance at 240 nm along with concomitant inactivation of the enzyme and reactivation of the modified enzyme by hydroxylamine. These results strongly suggest the presence of histidine residue(s) at or near the active site of the enzyme.

Amino Acid Sequence↗

PubMed2001

Mechanistic investigation of UDP-galactopyranose mutase from Escherichia coli using 2- and 3-fluorinated UDP-galactofuranose as probes.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 microM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of the 2-F substituent that is immediately adjacent to the anomeric center, and is consistent with a mechanism involving formation of oxocarbenium intermediates or transition states during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed, and irreversible with a K(I) of 270 microM and a k(inact) of 0.19 min(-1). Since the K(I) value is similar to Km, the observed inactivation is unlikely a result of tight binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase.

Bacterial Proteins↗

PubMed2003

Effect of thiohydroxyl compounds on tyrosinase: inactivation and reactivation study.

An unusual thioether bridge (Cys-His) has been detected at the active site of mushroom tyrosinase, and the effects of thiohydroxyl compounds such as dithiothreitol (DTT) and beta-mercaptoethanol (beta-ME) on Cu2+ at the active site have been elucidated. Treatment with DTT and beta-ME on mushroom tyrosinase completely inactivated 3,4-dihydroxyphenylalanine oxidase activity in a dose-dependent manner. Sequential kinetic studies revealed that DTT and beta-ME caused different mixed-type inhibition mechanisms: the slope-parabolic competitive inhibition (Ki = 0.143 mM) by DTT and slope-hyperbolic noncompetitive inhibition (Ki = 0.0128 mM) by beta-ME, respectively. Kinetic Scatchard analysis consistently showed that mushroom tyrosinase had multiple binding sites for DTT and beta-ME with different affinities. Reactivation study of inactivated enzyme by addition of Cu2+ confirmed that DTT and beta-ME directly bound with Cu2+ at the active site. Our results may provide useful information regarding interactions of tyrosinase inhibitor for designing an effective whitening agent targeted to the tyrosinase active site.

Agaricales↗

PubMed1971

Rat liver alcohol dehydrogenase. Purification and properties.

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.

Alcohol Oxidoreductases↗

PubMed1998

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme.

The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

Animals↗

PubMed1976

[On the histotopochemistry of the uterovaginal region in the quail (Coturnix coturnix japonica) (author's transl)].

The surface epithelium of vagina, uterovaginal region and uterus as well as the uterine and uterovaginal glands of 18 mature female quails were studied with histochemical methods. As in other avian species also in the quail a storage of spermatozoa in the lightly coiled uterovaginal glands takes place. The functional specialization of these glands is underlined by their distinct enzyme pattern. A strong reactivity of enzymes from oxidative pathways and of adenosine triphosphatase between epithelium and glandular luminal content. Alkaline phosphatase in the glandular epithelium was observed only when an egg is transported through the uterovaginal region. As in other vertebrate sperm storing sites also in the uterovaginal region of the quail the presence of a strong steroid dehydrogenase activity is registered.

Adenosine Triphosphatases↗

PubMed1984

Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients.

Purified RNA polymerase I was phosphorylated by the endogenous protein kinase or dephosphorylated by alkaline phosphatase and used as antigen in a radioimmunoassay with sera from systemic lupus erythematosus patients or serum from an immunized rabbit. Enzyme incubated in the absence of ATP or phosphatase served as control. Three to seven times more of the autoantibodies in the patients' sera reacted with phosphorylated RNA polymerase I than with control enzyme. The reactivity of the dephosphorylated enzyme with lupus autoantibodies was only 50-60% of that observed with control enzyme. Neither phosphorylation nor dephosphorylation of the enzyme had an effect on its reaction with the rabbit antibodies. The effect of phosphorylation on the reaction of each RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients' antibodies was determined by an immunoblot procedure following resolution of the subunits on polyacrylamide gels. Prior phosphorylation of the enzyme resulted in a dramatic increase in binding of each patient's antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control enzyme. Strikingly, antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5 antibodies in the serum of one patient were only detected with phosphorylated RNA polymerase I. The present data suggest that at least a significant fraction of the anti-RNA polymerase I autoantibodies in the sera of systemic lupus erythematosus patients might be directed against phosphorylated sites on the enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.

Adenosine Triphosphate↗

PubMed1980

Evidence of inactivation of pineal indoleamine N-acetyltransferase by protein thiol:disulfide exchange.

Pineal indoleamine N-acetyltransferase activity in homogenates is rapidly reduced at pH 6.8 by treatment with cystamine or arginine vasotocin. Other disulfides including glutathione disulfide, penicillamine disulfide, and N,N'-diacetylcystamine are either ineffective or less effective. The diamine analogous to cystamine, diaminohexane, is also ineffective. Inactivation by cystamine is accelerated at higher pH and is temperature- and time-dependent. It was also found that cystamine treatment inactivated N-acetyltransferase in intact pineal cells. Treatment with dithiothreitol reactivated the cystamine- or arginine vasotocin-inactivated enzyme formed in broken cell preparations and the cystamine-inactivated enzyme formed in intact cells. These observations indicate that pineal N-acetyltransferase can be inactivated by protein thiol:disulfide exchange; further research is required to ascertain whether this mechanism is of physiological significance.

Acetyltransferases↗

PubMed2004

The use of model systems to study biological functions of Nox/Duox enzymes.

ROS (reactive oxygen species; including superoxide and H202) are conventionally thought of as being broadly reactive and cytotoxic. Phagocytes utilize an NADPH oxidase to generate large amounts of ROS, and exploit their toxic properties as a host-defence mechanism to kill invading microbes. However, the recent discovery of the Nox and Duox enzymes that are expressed in many non-phagocytic cells implies that the 'deliberate' generation of ROS has additional cellular roles, which are currently incompletely understood. Functions of ROS in mammals have been inferred primarily from cell-culture experiments, and include signalling for mitogenic growth, apoptosis and angiogenesis. Nox/Duox enzymes may also provide H202 as a substrate for peroxidase enzymes (or, in the case of Duox, for its own peroxidase domain), thereby supporting peroxidative reactions. A broad comparison of biological functions of ROS and Nox enzymes across species and kingdoms provides insights into possible functions in mammals. To further understand novel biological roles for Nox/Duox enzymes, we are manipulating the expression of Nox/Duox enzymes in model organisms including Caenorhabditis elegans, Drosophila melanogaster and mouse. This chapter focuses on new insights into the roles of Nox enzymes gained from these approaches.

Animals↗

PubMed1984

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase.

Goat antisera against (Na+ + K+)-ATPase and its isolated subunits and against (K+ + H+)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na+ + K+)-ATPase antisera cross-reacted with (K+ + H+)-ATPase or inhibited its enzyme activity. The same was true for the (K+ + H+)-ATPase antiserum with regard to (Na+ + K+)-ATPase and its subunits and its enzyme activity. So notwithstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.

Adenosine Triphosphatases↗

PubMed1984

An enzyme histochemical study of the sinuses of reactive lymph nodes.

The enzyme histochemistry of the cells lining and within the marginal and medullary sinuses of twenty human reactive lymph nodes has been studied. The sinuses contain luminal ('reticular') cells which are strongly positive for certain hydrolytic enzymes, including acid-alpha-naphthyl acetate esterase, acid phosphatase and beta-glucuronidase. In addition, the lining ('littoral') cells on both sides of the medullary sinuses are positive for these enzymes. In contrast, enzyme-containing lining ('littoral') cells of the marginal (subcapsular) sinuses are observed only on the inner aspect of the sinuses, the outer aspect being negative. Alkaline phosphatase is not present in the sinusoidal cells but 5'-nucleotidase is seen in varying amounts. These findings are supported by an ultrastructural study of three of the nodes, using a staining method for esterase activity. The different enzyme histochemical properties of the littoral cells in the marginal and medullary sinuses closely mirrors that observed when, for example, these structures are stained immunohistochemically for IgA or J chain.

5'-Nucleotidase↗

PubMed1977

Cross reactivity of rabbit antibodies against purified animal enzymes. Comparison of enzymes of human and animal origin, II.

Enzyme preparations used for quality control of enzyme activity determinations in clinical chemistry should be very similar to human enzymes. Antibodies against e.g. porcine enzymes show cross reactivity with the corresponding human enzymes, indicating partial identity of the enzyme proteins. As the enzyme-antibody precipitates retain catalytic activity the similarity between the enzyme proteins is not limited to the catalytic sites.

Animals↗

PubMed1981

Molecular symmetry and metastable states of enzymes exhibiting half-of-the-sites reactivity.

Half-of-the-sites reactivity in oligomeric enzymes has generally been accepted as evidence for structural asymmetry between subunits. However, we show that the symmetric two-state allosteric model [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] is quantitatively consistent with half-of-the-sites reactivity data for several hexameric and tetrameric enzymes. Specifically, the time courses for both the modification and the inactivation of glutamate dehydrogenase by glutamyl alpha-chloromethyl ketone and uridine diphosphoglucose dehydrogenase by 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid are fit with just five parameters for each enzyme-modifier pair. In the case of glyceraldehyde-3-phosphate dehydrogenase, the time courses for modification of the yeast enzyme by iodoacetic acid and the rabbit-muscle enzyme by 3,3,3-trifluorobromoacetone are fit with the same model, and parameter values from these fits are used to generate theoretical inactivation curves which are found to agree well with the experimentally measured inactivation. We conclude that half-of-the-sites reactivity, if it is not an artifact of residual heterogeneity, could be a kinetic phenomenon related to metastability of partially modified states of a symmetric oligomer and that asymmetry between subunits should therefore not necessarily be inferred from such behavior. If similar metastability occurs in substrate binding, it may play a significant role in mechanism of catalysis and control. In such cases, the virtual inaccessibility of the substrate binding equilibrium would preclude conventional quasi-equilibrium models for the enzyme kinetics.

Animals↗

PubMed1994

On hypervariability at the reactive center of proteolytic enzymes and their inhibitors.

The pattern of synonymous and nonsynonymous substitutions at the reactive center of proteases (kallikrein) and their inhibitors (alpha 1-antitrypsin and serpin) was examined. In the case of alpha 1-antitrypsin, the proportion of different nonsynonymous sites exceeds that of different synonymous sites at the reactive center for sequence pairs of recent duplication. The result indicates that the positive selection has operated after duplication to increase functional diversity. In the cases of kallikrein, serpin, and remote sequence pairs of alpha 1-antitrypsin, the proportion of different synonymous sites at the reactive center exceeds that of different synonymous sites at the remaining region. The result indicates that gene conversion followed by natural selection is working. On the whole, it is concluded that hypervariability of amino acids at the reactive center is generated by an interaction among natural selection, random genetic drift, point mutation, and gene conversion. Gene duplication may provide potential for them to interact.

Animals↗

PubMed1995

Elevated thiobarbituric acid-reactive substances and antioxidant enzyme activity in the brain in Alzheimer's disease.

We determined levels of thiobarbituric acid-reactive substances (TBARS), a measure of lipid peroxidation, and the activity of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), and catalase (CAT) in the amygdala, hippocampus, pyriform cortex, superior and middle temporal gyri, inferior parietal lobule, middle frontal gyrus, occipital pole, and cerebellum of 13 Alzheimer's disease (AD) and 10 control brains. Levels of TBARS were elevated in all AD brain regions except the middle frontal gyrus, and elevation levels reached statistical significance in the hippocampus and pyriform cortex and marginal significance in the amygdala of AD subjects compared with age-matched controls. Significant elevation of GSH-Px activity was present in AD hippocampus compared with control. Moderate but statistically insignificant elevations of GSH-Px activity also were present in the amygdala and pyriform cortex in AD. GSSG-R activity was significantly elevated in the amygdala and hippocampus in AD subjects compared with controls. CAT activity was significantly elevated in AD hippocampus and superior and middle temporal gyri. SOD levels were elevated in all brain regions in AD patients compared with controls, although none of these elevations reached statistical significance. Antioxidant enzyme activities were significantly elevated where lipid peroxidation was most pronounced, suggesting a compensatory rise in antioxidant activity in response to increased free radical formation. This study supports the concept that the brain in AD is under increased oxidative stress and demonstrates that the oxidative changes are most pronounced in the medial temporal lobe, where histopathologic alterations are most severe.

Aged↗

PubMed2004

Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase.

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

Animals↗

PubMed1983

Histamine release into tracheal lumen and bronchial reactivity. The histamine liberation of proteolytic enzymes in the tracheal lumen in relation to the bronchomotoric reactivity.

The influence of tracheal lavage in a restricted area, with a proteolytic enzyme (Pronase) on the bronchial response to challenge with ACH and AE was studied in 15 dogs. Histamine concentrations measured in the tracheal liquid after lavage with Pronase were smaller than the values after tracheal lavage with allergen. Airway response to ACH inhalation was significantly increased after tracheal lavage with Pronase. Effects of proteolytic enzymes and allergens on the tracheal wall are compared. Allergens and proteolytic enzymes evidence a different mode of action in increasing reactivity of peripheral airways.

Allergens↗