Purification and kinetic evaluation of polyamine oxidase from Avena sativa L

MAIALE SANTIAGO; RUIZ OSCAR

villa Carlos Paz, Cordoba

Congreso; 28 Reunion anual de la sociedad argentina de investigaciones en bioquimicas y biologia molecular; 2002

Sociedad Argentina en Investigaciones en Bioquimica y Biologia Molecular

Polyamines (PA) show ubiquitous occurrence in prokaryotic and eukaryotic organisms. The PA spermidine (SPD) and spermine (SPM) and the related diamines, putrescine and cadaverine; appear to play an important role in processes of cell growth, division and differentiation. PA catabolism in plant is associated with physiological events such as lignifications, cell wall stiffening and cellular defense. Enzymes involved in PA catabolism are formally classified as copper-containing amine oxidase and flavin-containing polyamine oxidase. In comparison with large and detailed investigation focused on copper amine oxidase, only little attention has been devoted to study flavoprotein polyamine oxidase (PAOs). Flavin-containing PAOs have been isolated particularly from plants belonging to the Gramineae. We purified to homogeneity (PAGE-SDS-Coomansie Blue) the PAO from primary leaves of Avena sativa. The crude enzymatic preparation was obtained by vacuum infiltration and precipitation with acetone, forwarder by DEAE and molecular filtration technique in FPLC. The routinely enzymatic activity was assayed with a peroxidase-guaiacol coupled reaction. Additionally we used radioactive substrates in order to evaluate differential activity on SPD and SPM. No activity was found on putrescine, cadaverine and hexanediamine, but these diamines were good inhibitors. The diaminoguanidine was also an inhibitor and double reciprocal graphic was made using SPD and SPM as substrates. Surprisingly 1, 3 diaminopropane (DAP) didn’t inhibit but 1-aminooxy 3-aminopropane (APA) – analogous of DAP was an effective inhibitor.