title: Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies.
abstract: AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k(cat) values as high as 7 s(-1). Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k(cat)/K(m) values between 10(2) and 10(4) M(-1) s(-1). K(m)(O(2)) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k(cat)/K(m) values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell K(m) values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k(cat)/K(m) have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California, United States of America.
authors.forename: Sam H
authors.initials: SH
authors.lastname: Shen
authors.forename: Diana L
authors.initials: DL
authors.lastname: Wertz
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001419:Bacteria / Q000378:metabolism; D018407:Bacterial Physiological Phenomena / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D004330:Drosophila; D004789:Enzyme Activation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007700:Kinetics; D051379:Mice; D009765:Obesity / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D014162:Transfection
chemical_list: D015815:Cell Adhesion Molecules; D006631:Amine Oxidase (Copper-Containing); C492678:semicarbazide-sensitive amine oxidase-vascular adhesion protein-1, mouse