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Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

Nonylphenol (NP) and octylphenol (OP) are pervasive environmental contaminants belonging to the broader class of compounds known as alkylphenols, with potential human toxic effects. Classified as “xenoestrogens,” NP and OP are able to interfere with the cell endocrine physiology via a direct interaction with the estrogen receptors. Here, using HepG2 cells in culture, the changes of the cell redox balance and mitochondrial activity induced by OP and NP have been investigated atμM concentrations, largely below those provoking acute toxicity, as those typical of environmental contaminants. Following 24 h cell exposure to both OP and NP, ROS production appeared significantly increased (p≤0.01), together with the production of higher NO oxides (p=0.003) and peroxynitrated protein-derivatives (NP versus CTR,p=0.003). The mitochondrial proton electrochemical potential gradient instead was decreased (p≤0.05), as the oxygen consumption by complex IV, particularly following incubation with NP (NP versus CTR,p=0.017). Consistently, the RT-PCR and Western blot analyses proved that the OP and NP can modulate to a different extent the expression of the inducible NOS (NP versus CTR,p≤0.01) and the endothelial NOS (OP versus CTR,p≤0.05), with a significant variation of the coupling efficiency of the latter (NP versus CTR,p≤0.05), a finding that may provide a novel clue to understand the specific xenoestrogenic properties of OP and NP.