ABSTRACT
Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Fabaceae/chemistry , Pisum sativum/chemistry , Plants, Medicinal , Amine Oxidase (Copper-Containing)/isolation & purification , Animals , Antibodies , Binding, Competitive , Blotting, Western , Hydrogen-Ion Concentration , Immunodiffusion , Isoelectric Focusing , Kinetics , RabbitsABSTRACT
The interaction of pea seedling amine oxidase with cytokinins was examined to probe a possible connection between cytokinin oxidase and amine oxidase by determining whether cytokinins are substrates or inhibitors of the latter. Kinetic measurements suggest that cytokinins are weak competitive inhibitors of amine oxidase while their behaviour as substrates was not observed. The absence of enzymatic activity with cytokinins as substrates denies the identity or even any similarity of these two enzymes which was previously considered [Hare, P.D. and van Staden, J. (1994) J. Physiol. Plant., 91, 128]. From the values of the inhibition constants obtained it seems unlikely that cytokinins take part in the regulation of amine oxidase activity in vivo. Their inhibitory effect on amine oxidase may be similar to that of some alkaloids studied earlier.
Subject(s)
Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Cytokinins/pharmacology , Plant Proteins/antagonists & inhibitors , Amine Oxidase (Copper-Containing)/metabolism , Binding, Competitive , Cytokinins/metabolism , Kinetics , Pisum sativum/enzymology , Plant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Kinetic properties of novel amine oxidases isolated from a mold Aspergillus niger AKU 3302 were compared to those of typical plant amine oxidase from pea seedling (EC 1.4.3.6). Pea amine oxidase showed highest affinity with diamines, such as putrescine and cadaverine, while fungal enzymes oxidized preferably n-hexylamine and tyramine. All enzymes were inhibited by carbonyl reagents, copper chelating agents, some substrate analogs and alkaloids, but there were quite significant differences in the sensitivity and inhibition modes. Aminoguanidine, which strongly inhibited pea amine oxidases showed only little effect on fungal enzymes. Substrate analogs such as 1.5-diamino-3-pentanone and 1-amino-3-phenyl-3-propanone, which were potent competitive inhibitors of pea amine oxidases, inhibited fungal enzymes much more weakly and non competitively. Also various alkaloids behaving as competitive inhibitors of pea amine oxidase inhibited the fungal enzymes non competitively. Very surprising was the potent inhibition of fungal enzymes by artificial substrates of pea amine oxidases, E- and Z-1,4-diamino-2-butene. The relationships between the different inhibition modes and possible binding at the active site are discussed.
