Monoamine oxidase A and B ( MAO A and B) play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. MAO also acts on xenobiotics, including oxidation of the parkinsonism-producing neurotoxin MPTP. Furthermore, alterations in the structure, expression or function of these enzymes have been suspected in some psychiatric disorders, such as depression and schizophrenia. The objective of this proposal is to characterize further MAO A and B at the biochemical and molecular levels. These studies include determination of how flavin adenine dinucleotide (FAD) is covalently linked to the MAO polypeptide, examination of the functional domains in expression systems and determination of how MAO is anchored to the mitochondrial membrane, and cloning the MAO A and B genes to determine their nucleotide sequences, including the 5'-upstream region for the promotor and regulatory elements. Selected peptides (that correspond to the domain in MAO that is known to bind FAD) and bacterial MAO-fusion proteins will be tested as substrates for the covalent coupling of riboflavin or its derivatives. A putative flavin-coupling enzyme(s) will be isolated and characterized, and the substrate specificity for flavin coupling will be investigated by altering the amino acid sequence around the FAD-binding site using site-directed mutagenesis and by preparing synthetic peptides with selected amino acid substitutions. The domain(s) that is anchored to the membrane will be identified by proteolytic cleavage of MAO-containing liposomes followed by identification of the external peptides. The structure/activity relationships of functional domains will be further elucidated using a mammalian expression system. MAO positive clones have recently been identified from a human genomic library using cDNAs that encode MAO A and B. These clones will be restriction analyzed and subcloned for sequencing studies while further screening for more positive clones are underway. Emphasis will be placed upon comparing the 51-upstream promotor and regulatory sequences to gain insight into what elements control the preferential expression of these two enzymes in different subsets of neurons in various regions of brain.
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