The uptake of spermine into mammalian mitochondria indicated the need to identify its catabolic pathway in these organelles. Bovine liver mitochondria were therefore purified and their capacity for natural polyamine uptake was verified. A kinetic approach was then used to determine the presence of an MDL 72527-sensitive enzyme with spermine oxidase activity in the matrix of bovine liver mitochondria. Western blot analysis of mitochondrial fractions and immunogold electron microscopy observations of purified mitochondria unequivocally confirmed the presence of a protein recognized by anti-spermine oxidase antibodies in the mitochondrial matrix. Preliminary kinetic characterization showed that spermine is the preferred substrate of this enzyme; lower activity was detected with spermidine and acetylated polyamines. Catalytic efficiency comparable to that of spermine was also found for 1-aminododecane. The considerable effect of ionic strength on the Vmax/KM ratio suggested the presence of more than one negatively charged zone inside the active site cavity of this mitochondrial enzyme, which is probably involved in the docking of positively charged substrates. These findings indicate that the bovine liver mitochondrial matrix contains an enzyme belonging to the spermine oxidase class. Because H2O2 is generated by spermine oxidase activity, the possible involvement of the latter as an important signaling transducer under both physiological and pathological conditions should be considered.

Department of Biomedical Sciences University of Padova Via G Colombo 3 35131 Padova Italy

Department of Comparative Biomedicine and Food Science Polo Agripolis Viale dell'Università 16 University of Padova 35020 Legnaro Italy; Regional Centre for Advanced Technologies and Materials Department of Physical Chemistry Palacky University in Olomouc 17 Listopadu 1192 12 771 46 Olomouc Czech Republic

Department of Molecular Medicine University of Padova Via G Colombo 3 35131 Padova Italy

Department of Molecular Medicine University of Padova Via G Colombo 3 35131 Padova Italy; Consorzio Interuniversitario Istituto Nazionale Biostrutture e Biosistemi Viale delle medaglie d'Oro 305 00136 Roma Italy

Department of Molecular Science and Nanosystems Università Ca' Foscari Dorsoduro 2137 30123 Venezia Italy

Department of Technology and Health Italian Institute of Health 00161 Roma Italy

Istituto Pasteur Fondazione Cenci Bolognetti Department of Biochemical Sciences A Rossi Fanelli Sapienza University of Rome and Institute of Biology and Molecular Pathology Italian Research Council P le Aldo Moro 5 00185 Roma Italy

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