Methylamine and benzylamine induced hypophagia in mice: modulation by semicarbazide-sensitive benzylamine oxidase inhibitors and aODN towards Kv1.1 channels. (1/18)

1. In starved mice, the anorectic activity of methylamine (MET) and benzylamine (BZ), both substrates of semicarbazide-sensitive benzylamine oxidases (Bz-SSAO), was compared with that of the potassium channel blocking agents charybdotoxin (ChTX), tetraethylammonium (TEA), gliquidone (GLI), ammonium chloride (NH(4)(+)) and of the anoressants amphetamine (AMPH) and nicotine (NIC). After i.c.v. administration, an approximate ranking order of potency was: ChTX> or =AMPH>NIC=TEA> or =GLI> or =MET>BZ>NH(4)(+). 2. Clorgyline (2.5 mg kg(-1) i.p.) or deprenyl (10 mg kg(-1) i.p.) potentiated the anorectic effect of i.c.v.-administered BZ, NIC and AMPH. The effect of TEA was increased only by deprenyl, while MET, NH(4)(+), ChTX and GLI were not affected by either of the inhibitors. 3. The Bz-SSAO inhibitors alpha-aminoguanidine (50 mg kg(-1) i.p.), B24 (100 mg kg(-1) i.p.) and MDL 72274 (2.5 mg kg(-1) i.p.) potentiated the effect of i.p., but not of i.c.v.-administered MET. 4. Antisense oligodeoxyribonucleotides (aODN) to Kv1.1 potassium channels abolished the effect of BZ and TEA, but was ineffective in reducing the activity of MET and other compounds. 5. These results suggest that MET is endowed with peculiar hypophagic effects at dosage levels that are not able to affect gross behaviour in mice. The effect of MET, differently from BZ, seems unrelated to an increase in the central release of monoaminergic mediators, as well as to a Kv1.1 blocking activity. Through a reduction of the endogenous breakdown of MET, Bz-SSAO inhibitors enhance the central pharmacological activity of this amine.  (+info)

Active-sitve titration of pig plasma benzylamine oxidase with phenylhydrazine. (2/18)

Pig plasma benzylamine oxidase is a protein containing cupric copper and pyridoxal phosphate. The pyridoxal phosphate is stably linked to the enzyme. Discrepancies in the numbers of active sites per molecule of enzyme are reported in the literature. This paper shows that the fully active pure enzyme contains 3 mol of pyridoxal phosphate per mol, whereas enzymes with a lower specific activity are shown by titration with phenylhydrazine to have a lower pyrdoxal phosphate content.  (+info)

The action of plasma amine oxidase on beta-haloamines. Evidence for proton abstraction in the oxidative reaction. (3/18)

The action of plasma amine oxidase upon beta-Br-ethylamine beta-Cl-ethylamine, beta-OH-phenylethylamine, and beta-Cl-phenylethylamine was examined. Beta-Br-ethylamine is a substrate and irreversible inactivator of the enzyme. The enzyme becomes covalently labeled by the inactivator. Approximately 2 mol of inactivator are incorporated per mol of enzyme (MW 170,000). The reduced enzyme is not inactivated. The enzyme catalyzes the elimination of HCl from beta-Cl-phenylethylamine to produce phenylacetaldehyde. The rate of the elimination reaction is comparable to the normal oxidative reaction. We conclude that the occurrence of this elimination reaction establishes the ability of the enzyme to catalyze proton abstraction from C-1 of the substrate and that proton abstraction occurs during the catalytic oxidation normally catalyzed by plasma amine oxidase. Beta-Cl-ethylamine is only oxidized to corresponding aldehyde. Beta-OH-phenylethylamine is neither oxidized, nor does elimination occur. It is a competitive inhibitor in the oxidation of benzylamine and in the elimination of HCl from beta-Cl-phenylethylamine.  (+info)

Kinetic isotope effects on the catalytic activity of pig-plasma benzylamine oxidase. (4/18)

1. Isotope effects on the catalytic activity of benzylamine oxidase at pH 7 and 9 have been studied by steady-state and transient-state kinetics methods, using [alpha,alpha-2H]benzylamine as the substrate. 2. Replacement of the alpha-hydrogen atoms in benzylamine by deuterium has no significant effect on substrate-binding to benzylamine oxidase, neither does it affect the rate of reoxidation of the reduced form of the enzyme. Conversion of the primarily formed enzyme-substrate complex into the reduced enzyme species, however, exhibits an isotope effect of about 3. 3. The data obtained are consistent with a mechanism in which reduction of benzylamine oxidase takes place by a rapid pre-equilibration between enzyme and substrate to form an amine-pyridoxal Schiff-base, which is then tautomerized by a comparatively slow prototropic shift to an amino aldehyde-pyridoxamine Schiff-base from which there is a rapid hydrolytic release of the aldehyde product corresponding to the amine substrate. Proton abstraction from the alpha-carbon of the amine moiety in the primary Schiff-base appears to be at least partially rate-limiting for the tautomerization step, and hence for the entire process of enzyme reduction.  (+info)

Stopped-flow spectrophotometric characterization of enzymic reaction intermediates in the anaerobic reduction of pig-plasma benzylamine oxidase by amine substrates. (5/18)

Reduction of benzylamine oxidase by p-methoxybenzylamine under anaerobic conditions leads to biphasic absorbance changes at 470 nm. These reflect the intermediate formation of an enzyme substrate complex with spectral properties different from those of native enzyme and fully reduced enzyme. The spectrally modified enzyme-substrate complex exhibits a broad difference absorption band centered around 360 nm. The transient accumulation of this intermediate during reaction can be conveniently followed by stopped-flow techniques at wavelengths between 320 and 360 nm, where contributions from the subsequent reduction of the enzymic 470-nm chromophore are of minor significance. 2. Analogous intermediates exhibiting similar absorption spectra seem to be formed on reduction of the enzyme by benzylamine and other amine substrates which were tested. Substitution of benzylamine as the reducing substrate by [alpha, alpha-2H]benzylamine results in a decreased accumulation of the spectrally modified intermediate. This indicates that its formation is preceded by deprotonation of the alpha-carbon of the amine substrate. 3. Circular dichroism spectra of benzylamine oxidase exhibit a positive band at 360 nm, lending support to the previous conclusion that benzylamine oxidase is a pyridoxal enzyme. Formation of the spectrally modified enzyme-substrate complex then most likely reflects the prototropic shift converting an amine-pyridoxal Schiff-base obtained by rapid pre-equilibration between enzyme and substrate into an aldehyde-pyridoxamine Schiff-base.  (+info)

Long-term high copper intake: effects on indexes of copper status, antioxidant status, and immune function in young men. (6/18)

BACKGROUND: Short-term high copper intake does not appear to affect indexes of copper status or functions related to copper status, but the effects of long-term high copper intake are unknown. OBJECTIVE: A study was conducted in men to determine the effect of long-term high copper intake on indexes of copper status, oxidant damage, and immune function. DESIGN: Nine men were confined to a metabolic research unit (MRU) for 18 d and were fed a 3-d rotating menu providing an average of 1.6 mg Cu/d. The men continued the study under free-living conditions for 129 d and supplemented their usual diets with 7 mg Cu/d. The men then returned to the MRU for 18 d of the same diet as during the first period, except that copper intake was 7.8 mg/d. Plasma copper, ceruloplasmin activity, ceruloplasmin protein, plasma malondialdehyde, benzylamine oxidase activity, erythrocyte superoxide dismutase, hair copper, urinary copper, and urinary thiobarbituric acid-reactive substances were measured during each MRU period. RESULTS: Ceruloplasmin activity, benzylamine oxidase, and superoxide dismutase were significantly higher at the end of the second MRU period than at the end of the first. Urinary copper excretion, hair copper concentrations, and urinary thiobarbituric acid-reactive substances were significantly higher during the second MRU period than during the first. Polymorphonuclear cell count, the percentage of white blood cells, lymphocyte count, and interleukin 2R were affected by copper supplementation. Antibody titer for the Beijing strain of influenza virus was significantly lower in supplemented subjects after immunization than in unsupplemented control subjects. CONCLUSIONS: Under highly controlled conditions, long-term high copper intake results in increases in some indexes of copper status, alters an index of oxidant stress, and affects several indexes of immune function. The physiologic implications of these changes are unknown.  (+info)

The kinetics of reoxidation of reduced benzylamine oxidase. (7/18)

1. The mechanism of reoxidation of reduced benzylamine oxidase has been investigated at different pH between 6 and 10 by steady-state and transient-state kinetic methods. 2. The reoxidation process involves minimally a second-order interaction between reduced enzyme and oxygen leading to the formation of a spectrally modified enzyme intermediate, and a subsequent first-order step converting this intermediate into free enzyme. The variation with pH of rate constants according to such a reaction scheme is reported. 3. Under aerobic conditions the oxygen-independent reaction represents the main rate-limiting step in the catalytic process at alkaline pH. At neutral or acid pH the interaction between reduced enzyme and oxygen becomes mainly rate-limiting, indicating that the concentration of oxygen may be a critical factor controlling enzyme activity under physiological conditions. 4. The spectrally modified intermediate formed during the reoxidation process exhibits a difference-absorption band centered around 290 nm in comparison to free enzyme, and an additional difference-absorption band at 470 nm in comparison to reduced enzyme. These data indicate that formation of the intermediate, besides leading to a reappearance of the 470-nm absorption band disappearing on reduction of the enzyme, results in a spectral perturbation of one or several aromatic amino-acid residues in the protein. This perturbation could possibly reflect a conformational change of the enzymes.  (+info)

Properties of cupric ions in benzylamine oxidase from pig plasma as studied by magnetic-resonance and kinetic methods. (8/18)

Benzylamine oxidase from pig plasma has been studied by a variety of chemical and physical techniques. 1. Analytical ultracentrifugation, gel electrophoresis and isoelectric-focusing studies suggest that the enzyme is composed of two subunits with closely similar primary structures. 2. E.s.r. and n.m.r. measurements show that the enzyme contains two well-separated (greater than 0.6 nm) Cu2+ ions at chemically distinct sites. Each Cu2+ ion is coordinated by two water molecules, one 'axial' and the other 'equatorial'. Both water molecules undergo fast exchange (10(5)--10(8) s-1) with solvent and are deprotonated in the pH range 8--9, but only the equatorial water molecule is displaced by the inhibitors N3- and CN-. 3. Kinetic and e.s.r. measurements show that azide and cyanide compete against O2 binding and also make the two Cu2+ sites identical. It is concluded that Cu2+ must participate in the re-oxidation of reduced enzyme by molecular O2.  (+info)

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