Characterization of polyamine oxidase from the aquatic nitrogen-fixing fern Azolla imbricata

Biochemical properties of a polyamine oxidase (PAO; EC 1.5.3.3) purified from the aquatic nitrogen-fixing fern Azolla imbricata (Roxb.) Nak. were studied. The native molecular mass of the enzyme estimated by Sephadex G 200 get filtration was 66.2 kDa. SDS-PAGE gave a single protein band corresponding to a molecular mass of 65.5 kDa. The light yellow enzyme had absorption maxima at 278, 372 and 454 nm with 1 mol FAD per mole enzyme molecule as its cofactor. The PAO was active on both the triamine Spd and the tetraamine Spm as substrates. However, it was inactive on the diamines Put and Cad. It had a pH optimum of 6.5 for both Spd and Spm. The K-m(S) for Spd and Spm were 6.71 x 10(-2) and 1.13 x 10(-1) nM, respectively. Pre-incubation with 10 mM of K+ (KCl), Ca2(+) (CaCl2) or Mg2+ (MgCl2) had no effect on PAO activity. However, 10 mM Cu2+ (CuCl2), Mn2+ (MnCl2) and Fe2+ (FeSO4) inhibited enzyme activity by 37%, 43% and 58%, respectively. The metal chelator EDTA (10 mM), the carbonyl reagent hydroxylamine (0.5 mM) and the sulfhydryl reagent p-chloro-mercuribenzoate (0.5 mM) had no effect on PAO activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

A novel tricopper(II) complex of a polyamine alcohol

The trinuclear copper(II) complex of a new polyamino alcohol ligand has been isolated; it exhibits a structure similar to that found at the active site of ascorbate oxidase.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Genetic covariation of neuroticism with monoamine oxidase activity and smoking

Variation in the personality trait of neuroticism is known to be affected by genetic influences, but despite a number of association studies, the genes involved have not yet been characterized. In a recent study of platelet monoamine oxidase in 1,551 twin subjects, we found a significant association between monoamine oxidase activity and scores on the Eysenck Personality Questionnaire neuroticism scale. Further analyses presented here indicate that both neuroticism and monoamine oxidase activity are associated with variation in smoking habits, and that adjusting for the effect of smoking strengthens the association between MAO and neuroticism. Analysis of the genetic and environmental sources of covariation between neuroticism, smoking, and monoamine oxidase activity show that approximately 8% of the genetic variance in neuroticism is due to the same additive genetic effects that contribute to variation in monoamine oxidase activity, suggesting that variation in neuroticism is associated in part with aspects of serotonin metabolism. (C) 2001 Wiley-Liss, Inc.

The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition

Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.

Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase

A metabolic flux model was developed for Streptococcus zooepidemicus to compare the metabolism of glucose and maltose during aerobic batch cultivation. Lactic acid was the main product of glucose metabolism whereas acetic acid was the main product of maltose metabolism. This difference was chiefly attributed to the two-fold higher flux through NADH oxidase in maltose-grown cells that enabled the ATP generation rate to remain high despite a slower maltose consumption rate. The two-fold higher flux was matched by a two-fold increase in NADH oxidase activity, 2.53 +/- 0.1 mumol NADH min(-1) mg(-1) protein on maltose versus 1.07 +/- 0.04 Rmol NADH min(-1) mg(-1) protein on glucose, indicating that NADH oxidase activity is regulated by the energy status of the cell. Surprisingly, the energy status of the cell had little impact on hyaluronic acid (HA) yield and molecular weight. (C) 2003 Elsevier Science B.V. All rights reserved.

Multi-centre Phase II trial of the polyamine synthesis inhibitor SAM486A (CGP48664) in patients with metastatic melanoma

Purpose: To determine the activity and tolerability of SAM496A, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), in patients with metastatic melanoma who had not received prior chemotherapy. Selected patients were offered participation in two sub-studies examining early changes in tumor metabolism with FDG-PET and changes in tumor polyamine content. Patients and methods: Fifteen patients with measurable metastatic melanoma, normal cardiac function, and no known CNS metastases were eligible and received SAM486A by 1-hour IV infusion daily for 5 days every 3 weeks. Response was assessed by SWOG criteria. Results: No patient had a confirmed partial response. Fatigue/lethargy, myalgia and neutropenia were the main toxicities but no febrile neutropenia or grade 4 non-hematological toxicity occurred. Five patients had PET scans pre-treatment and on days 8-12 of cycle 1. No patient had reduction of tumor metabolism. Serial biopsy in one patient showed alterations in polyamines consistent with SAMDC inhibition. Conclusions: Using the present dose and schedule of administration, SAM486A does not have significant therapeutic potential in patients with metastatic melanoma.

An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid

To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (K-I and k(inact)) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (K-I and k(inact)) for isoniazid were 48.6 mu M and 0.042 min(-1) and 79.3 mu M and 0.039 min(-1). Clorgyline was a selective inactivator of CYP1A2 (6.8 mu M and 0.15 min(-1)). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors.

Direct electrochemically driven catalysis of bovine milk xanthine oxidase

The complex molybdoenzyme xanthine oxidase (XO) catalyses the oxidation of xanthine to uric acid. Here we report the first direct (unmediated) catalytic electrochemistry of the enzyme in the presence of xanthine. The only non-turnover response (without substrate present) is a sharp two-electron wave from the FAD cofactor at -242 mV vs. NHE (pH 8.0). Upon addition of xanthine to the electrochemical cell a pronounced electrocatalytic anodic current appears at ca. +300 mV vs. NHE, but the FAD peak remains. This is unusual as the onset of catalysis should occur at the potential of the FAD cofactor (the site at which oxygen or NAD+ binds to the enzyme in solution). The observed electrochemical catalysis is prevented by the addition of known XO inhibitors allopurinol or cyanide. (c) 2005 Elsevier B.V. All rights reserved.

Protein film voltammetry of arsenite oxidase from the chemolithoautotrophic arsenite-oxidizing bacterium NT-26

The chemolithoautotrophic bacterium NT-26 (isolated from a gold mine in the Northern Territory of Australia) is unusual in that it acquires energy by oxidizing arsenite to arsenate while most other arsenic-oxidizing organisms perform this reaction as part of a detoxification mechanism against the potentially harmful arsenite [present as As(OH)(3) at neutral pH]. The enzyme that performs this reaction in NT-26 is the molybdoenzyme arsenite oxidase, and it has been previously isolated and characterized. Here we report the direct (unmediated) electrochemistry of NT-26 arsenite oxidase confined to the surface of a pyrolytic graphite working electrode. We have been able to demonstrate that the enzyme functions natively while adsorbed on the electrode where it displays stable and reproducible catalytic electrochemistry in the presence of arsenite. We report a pH dependence of the catalytic electrochemical potential of -33 mV/pH unit that is indicative of proton-coupled electron transfer. We also have performed catalytic voltammetry at a number of temperatures between 5 and 25 degrees C, and the catalytic current (proportional to the turnover number) follows simple Arrhenius behavior.

Chronic ethanol treatment leads to increased ornithine decarboxylase activity: Implications for a role of polyamines in ethanol dependence and withdrawal

Recent research has focused on the N-methyl-D-aspartate receptor system as a major site of ethanol action in the brain and specifically on compensatory changes in the expression of the polyamine-sensitive NR2B subunit. Therefore, we examined the effects of chronic ethanol treatment on polyamine homeostasis in the rat brain. Wistar rats were made dependent by ethanol vapor inhalation. This caused a rise in hippocampal ornithine decarboxylase (ODC) activity that was correlated with the appearance of physiological dependence. ODC activity returned to control levels within 3 days of ethanol withdrawal. Enzyme activity also increased in the cerebral cortex, striatum, and cerebellum of the ethanol-dependent rats. The concentration of the polyamines (putrescine, spermidine, and spermine) in the hippocampus was increased in ethanol-dependent rats. Injection of the ODC inhibitor, gamma-difluoromethylornithine (500 mg/kg) at the onset of withdrawal resulted in a significant reduction in the severity of withdrawal behaviors. The level of ODC activity and the severity of withdrawal behaviors were positively correlated. Perturbed polyamine homeostasis may represent an important molecular component in the initiation of ethanol withdrawal behaviors in the ethanol-dependent rat.

Ethanol inhibition of brain ornithine decarboxylase activity in the postnatal rat

The purpose of this study was to determine the relationship between ornithine decarboxylase activity (ODC; a marker for perturbed cell development), the blood alcohol level, and alcohol-induced microencephaly in the developing rat brain after binge treatment with ethanol vapour. By manipulating ethanol flow we were able to adjust vapour concentrations (24-65 mg ethanol/l air) such that an acute exposure of ethanol vapour for 3 h resulted in a range of blood alcohol levels (2.3-5.5 mg/ml). Acute studies showed that ethanol dose-dependently inhibited rat hippocampal and cerebellar ODC activity at PND4-PND10. There was a significant correlation between the blood alcohol level and degree of inhibition at all ages tested. Chronic treatment from PND4 to PND9 caused a significant decrease in both brain to body weight ratio and in hippocampal and cerebellar ODC activities at PND10. These results indicate that ethanol-induced disruption in ODC could play a significant role in ethanol's teratogenic effects during early postnatal development. (C) 1998 Elsevier Science Inc.

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [H-3]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and H-1 NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [H-3]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8,D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.