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A Giartosio E Agostinelli B Mondovi 《Biochemical and biophysical research communications》1988,154(1):66-72
Analysis of the thermal unfolding of bovine serum amine oxidase by differential scanning calorimetry reveals for the dimeric protein a four domain structure consisting of two sets of domains. Each set contains two domains of similar size. The two smaller domains, in contrast with the larger ones, greatly differ in thermostability. Removal of copper changes the calorimetric pattern dramatically. The findings confirm that the metal cofactor plays a structural role. Since the enzyme contains two copper atoms and only one titratable carbonyl group, the calorimetric pattern suggests that the difference in thermostability of the two small domains might be due to the presence of a single organic cofactor. 相似文献
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Amani M Moosavi-Movahedi AA Floris G Mura A Kurganov BI Ahmad F Saboury AA 《Biophysical chemistry》2007,125(2-3):254-259
Thermal denaturation of Euphorbia latex amine oxidase (ELAO) has been studied by enzymatic activity, circular dichroism and differential scanning calorimetry. Thermal denaturation of ELAO is shown to be an irreversible process. Checking the validity of two-state it really describes satisfactorily the thermal denaturation of ELAO. Based on this model we obtain the activation energy, parameter T(*) (the absolute temperature at which the rate constant of denaturation is equal to 1 min(-1)), and total enthalpy of ELAO denaturation. HPLC experiments show that the thermal denatured enzyme conserves its dimeric state. The N(2)-->kD(2) model for thermal denaturation of ELAO is proposed: where N(2) and D(2) are the native and denatured dimer, respectively. 相似文献
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Lunelli M Di Paolo ML Biadene M Calderone V Battistutta R Scarpa M Rigo A Zanotti G 《Journal of molecular biology》2005,346(4):991-1004
Copper-containing amine oxidase extracted from bovine serum (BSAO) was crystallized and its three-dimensional structure at 2.37A resolution is described. The biological unit of BSAO is a homodimer, formed by two monomers related to each other by a non-crystallographic 2-fold axis. Each monomer is composed of three domains, similar to those of other amine oxidases from lower species. The two monomers are structurally equivalent, despite some minor differences at the two active sites. A large funnel allows access of substrates to the active-site; another cavity, accessible to the solvent, is also present between the two monomers; this second cavity could allow the entrance of molecular oxygen necessary for the oxidative reaction. Some sugar residues, bound to Asn, were still present and visible in the electron density map, in spite of the exhaustive deglycosylation necessary to grow the crystals. The comparison of the BSAO structure with those of other resolved AO structures shows strong dissimilarities in the architecture and charge distribution of the cavities leading to the active-site, possibly explaining the differences in substrate specificity. 相似文献
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Agostinelli E Palmigiani P Vedova LD Tempera G Belli F Seiler N 《Biochemical and biophysical research communications》2006,340(3):840-844
MDL 72527 was considered a selective inhibitor of FAD-dependent polyamine oxidases. In the present communication, we demonstrate that MDL 72527 inactivates bovine serum amine oxidase, a copper-containing, TPQ-enzyme, time-dependently at 25 degrees C. In striking contrast, the enzyme remained active after incubation with excessive MDL 72527 at 37 degrees C, even after 70 h of incubation. Inactivation of BSAO with MDL 72527 at 25 degrees C did not involve the cofactor, as was shown by spectroscopy and by reaction with phenylhydrazine. Docking of MDL 72527 is difficult, owing to its size and two lipophilic moieties, and it has been shown that minor changes in reaction rate of substrates cause major changes in K(m) and k(cat)/K(m). We hypothesise that subtle conformational changes between 25 and 37 degrees C impair MDL 72527 from productive binding and prevent the nucleophilic group from reacting with the double bond system. 相似文献
7.
Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO
bovine serum amine oxidase
- DDC
N,N-diethyldithiocarbamate
- PQQ
pyrroloquinoline quinone 相似文献
8.
L Morpurgo E Agostinelli J Muccigrosso F Martini B Mondovi L Avigliano 《The Biochemical journal》1989,260(1):19-25
Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed. 相似文献
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Pietrangeli P Nocera S Fattibene P Wang X Mondovì B Morpurgo L 《Biochemical and biophysical research communications》2000,267(1):174-178
Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling. 相似文献
10.
Sharmin S Sakata K Kashiwagi K Ueda S Iwasaki S Shirahata A Igarashi K 《Biochemical and biophysical research communications》2001,282(1):228-235
The toxicity of extracellular spermine, determined in the presence of fetal calf serum, was studied using three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase in fetal calf serum produces aminodialdehyde generating acrolein spontaneously, H(2)O(2), and ammonia from spermine. Spermine toxicity was prevented by aldehyde dehydrogenase, but not by catalase. Similar concentrations of spermine and acrolein were needed to produce toxicity. Other aldehydes (formaldehyde, acetaldehyde, and propionaldehyde) and hydrogen peroxide were less toxic than acrolein. Spermidine and 3-aminopropanal, which produces acrolein, also exhibited severe cytotoxicity. The degree of cytotoxicity of spermine, spermidine, and 3-aminopropanal was nearly parallel with the amount of acrolein produced from each compound. Thus, it was deduced that acrolein is a major toxic compound produced from polyamines (spermine and spermidine) by amine oxidase. 相似文献
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Arancia G Calcabrini A Marra M Crateri P Artico M Martone A Martelli F Agostinelli E 《Amino acids》2004,26(3):273-282
Summary. Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H2O2 and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H2O2. However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells. 相似文献
12.
Roles of the two copper ions in bovine serum amine oxidase 总被引:3,自引:0,他引:3
With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
P. Turini S. Sabatini O. Befani F. Chimenti C. Casanova P.L. Riccio B. Mondovi 《Analytical biochemistry》1982,125(2):294-298
A new method for the purification of bovine plasma amine oxidase is described. The enzyme is purified by ammonium sulfate precipitation and by affinity chromatography performed with AH-Sepharose 4B and concanavalin A-Sepharose. Three activity peaks were separated, all showing similar properties. Specific activity is the highest described for this enzyme. The enzyme appears to contain 2 copper atoms and 1 carbonyl group/molecule. 相似文献
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Vibrational circular dichroism (VCD) spectroscopy has been used for the first time to investigate the thermal denaturation of proteins in H(2)O solutions. Films prepared from heated aqueous solutions were used for these investigations. A well-known alpha-helical protein, bovine serum albumin (BSA), is used for this first study. Both VCD and infrared absorption results obtained for BSA films indicate that the heat treatment of BSA induces significant amounts of beta-sheet structure and that the denaturation process is irreversible. To verify the irreversible nature of thermal denaturation, optical rotation was also measured as a function of temperature in both heating and cooling cycles. These results also indicate that thermal denaturation of BSA in solution is irreversible. This study establishes the usefulness of films for VCD investigations and offers new avenues for VCD studies on biologically important systems. 相似文献
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Spermine is a substrate of lentil (Lens culinaris) seedling amine oxidase and the oxidation products are reversible inactivators of the enzyme. The spermine is oxidized at the terminal amino groups to a dialdehyde: 2 moles of hydrogen peroxide and 2 moles of ammonia per mole of spermine are formed. The pH optimum of the enzyme with spermine is 7.9 in TI-HCI buffer; the Km value is 4.4·10−4 molar, similar to that found with other substrates (putrescine and spermidine). 相似文献
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B Mondovi L Morpurgo E Agostinelli O Befani J McCracken J Peisach 《European journal of biochemistry》1987,168(3):503-507
Electron spin-echo envelope modulation spectroscopy has been used to compare the local environment of Cu(II) in native bovine serum amine oxidase, containing two copper atoms/dimer molecule, and in a protein preparation, half depleted of copper, which has little enzymatic activity. For each preparation, two different populations of coordinated imidazoles with inequivalent magnetic coupling to copper could be recognized. In addition, water was shown to be a ligand to copper. No differences in coordinated ligand structures between the native and half-Cu-depleted preparations could be seen. In addition, the amount of ambient, non-coordinated water detected for native and half-Cu-depleted proteins was found to be nearly equivalent. However, the addition of phenylhydrazine, an inhibitor that binds to the pyrroloquinoline quinone cofactor but not to Cu(II) in the native enzyme, displaces ambient water near copper. 相似文献
18.
Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity
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In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR. 相似文献
19.
The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on kcat and kcat/Km parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (Kd). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O2 to H2O2. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of [14C]benzylamine and [3H]NaCNBH3. The use of the relatively weak reductant, NaCNBH3, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH3 in the presence of substrate, leading to the incorporation of 1 mol of [14C]benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with [3H]NaCNBH3, analogous to an earlier study with [3H]NaCNBH4 (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase. 相似文献