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PLoS One. 2012;7(1):e29270. doi: 10.1371/journal.pone.0029270. Epub 2012 Jan 4.

Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies.

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1
Department of Chemistry, University of California, Berkeley, California, United States of America.

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.

PMID:
22238597
PMCID:
PMC3251558
DOI:
10.1371/journal.pone.0029270
[Indexed for MEDLINE]
Free PMC Article
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