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This study evaluates the effects of a copper amine oxidase (histaminase) purified from the pea seedling as a free or immobilized enzyme on asthmalike reactions to inhaled antigen in actively sensitized guinea pig in vivo. Male albino guinea pigs, sensitized with ovalbumin, were challenged with the antigen given by aerosol; free histaminase or CNBr-Sepharose immobilized histaminase was given intraperitoneally (20 μg, 3 or 24 h before antigen challenge) or by aerosol (4 μg, 30 min before or during ovalbumin aerosol). The following parameters were examined: latency time for the onset of respiratory abnormalities, cough severity score, and occurrence and duration of dyspnea. We also evaluated lung histopathology, mast cell degranulation, and lung myeloperoxidase and malonydialdehyde levels. Histaminase significantly reduced the severity of cough and the occurrence of dyspnea and delayed the onset of respiratory abnormalities. Both enzymes prevented bronchial constriction, pulmonary air space inflation, leukocyte infiltration (evaluated as myeloperoxidase activity), and lipoperoxidation of cell membranes (evaluated as malonyldialdehyde production). No relevant differences in pharmacological potency were noted between free or immobilized enzyme. This study provides evidence that histaminase counteracts acute allergic asthmalike reaction in actively sensitized guinea pigs, raising the possibility of new therapeutic strategies for allergic asthma in humans.

For bovine serum amine oxidase, two different mechanisms of substrate-induced inactivation have been proposed. One consists of a slow oxidation by H2O2 of a conserved residue in the reduced enzyme after the fast turnover phase [Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovì, B.Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.267, 174-178] and the other of the oxidation by H2O2 of the dihydrobenzoxazole in equilibrium with the product Schiff base, during the catalytic cycle [Lee, Y., Shepard, E., Smith, J., Dooley, D.M.Sayre, L.M. (2001) Biochemistry40, 822-829]. To discriminate between the two mechanisms, the inactivation was studied using Lathyrus cicera (red vetchling) amine oxidase. This, in contrast to bovine serum amine oxidase, formed the Cu+-semiquinolamine radical with a characteristic UV-vis spectrum when oxygen was exhausted by an excess of any tested amine in a closed cuvette. The inactivation, lasting about 90 min, was simultaneous with the radical decay and with the formation of a broad band (shoulder) at 350 nm. No inactivation occurred when a thousand-fold excess of amine was rapidly oxidized in an L. cicera amine oxidase solution stirred in open air. Thus, the inactivation is a slow reaction of the reduced enzyme with H2O2, following the turnover phase. Catalase protected L. cicera amine oxidase from inactivation. This effect was substrate-dependent, varying from full protection (benzylamine) to no protection (putrescine). In the absence of H2O2, a specific inactivating reaction, without formation of the 350 nm band, was induced by some aldehydes, notably putrescine. Some mechanisms of inactivation are proposed.