flavin-adenine-dinucleotide and carbamylhydrazine

Upon induction with various amine sources, two different amine oxidases are expressed in the filamentous fungus Aspergillus niger. The enzymes which can be separated by anion exchange chromatography exhibit a similar substrate specificity pattern. From cofactor and inhibitor analysis it was found that one amine oxidase is identical to the earlier reported copper-containing amine oxidase (Yamada, H., Adachi, O. and Ogata, K. (1965) Agric. Biol. Chem. 29, 912-917) with 6-hydroxydopa (TOPA) quinone as the active site cofactor. The second form is a hitherto unknown flavoprotein of 55 kDa, which shows many of the characteristic properties of the mammalian monoamine oxidases (MAO). From substrate specificity and inhibitor susceptibility, it is suggested that the monoamine oxidase from A. niger (MAO-N) is a prototype of the two mammalian enzymes, MAO-A and MAO-B. A partial cDNA clone which encodes an amino-terminal peptide of 53 amino acid residues was identified by lambda gt11 immunoscreening. The consensus sequence of the putative flavin adenine dinucleotide (FAD) binding site is found within this sequence.

Topics: Amino Acid Sequence; Aspergillus niger; Binding Sites; Clorgyline; Consensus Sequence; Copper; Dihydroxyphenylalanine; Flavin-Adenine Dinucleotide; Molecular Sequence Data; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Pargyline; Recombinant Proteins; Semicarbazides; Sequence Homology; Spectrophotometry; Substrate Specificity

This experimental work tries to characterize the monoamine oxidase of microsomal origin through its kinetic and molecular properties, and to establish a comparative study with the enzyme present in rat liver mitochondria. The temperature effect upon this catalytic activity was examined and similar behaviour of MAO A and MAO B between both cellular fractions was found. The study of the pH dependence of initial velocity showed similar results both in mitochondria and in microsomes. The FAD cofactor is covalently attached to the MAO of microsomal origin. The FAD containing subunits corresponding to MAO A and MAO B, previous binding of the enzyme with [3H]pargyline and posterior SDS electrophoresis and fluorography, showed molecular weights of 65,900 and 62,400, respectively, in both cellular fractions. The inhibition curves with clorgyline, deprenyl, semicarbazide and KCN, measuring the remaining activity towards 1 microM of benzylamine, indicated that in mitochondria 5% of the total activity is due to the presence of SSAO activity whereas in microsomes this activity represents about 20%. From all these results it appears that mitochondrial and microsomal MAO are related enzymes, although further structural studies are necessary to confirm their possible identity.

Topics: Amine Oxidase (Copper-Containing); Animals; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Microsomes, Liver; Mitochondria, Liver; Monoamine Oxidase; Oxidoreductases Acting on CH-NH Group Donors; Rats; Rats, Inbred Strains; Semicarbazides; Temperature