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Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.

ABSTRACT: We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (K(m) 183 +/- 14 nM) and yet is metabolized fairly quickly (k(cat) 0.690 +/- 0.010 s(-1)), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorometric noncoupled assay that is 2 orders of magnitude more sensitive than the spectrophotometric benzylamine assay. The discovery of ANETA involved elaboration of the lead compound 6-methoxy-2-naphthalenemethaneamine by structure-based design, which recognized the ancillary cation binding site of BPAO as the most significant structural features controlling binding affinity. Structure-based design further ensured a high level of selectivity: ANETA is a good substrate of BPAO but is not a substrate of either porcine kidney diamine oxidase (pkDAO) or rat liver monoamine oxidase (MAO-B). ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond directly to the enzyme activity in real time.

SUBMITTER: Ling KQ

PROVIDER: S-EPMC2659760 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.

Ling Ke-Qing KQ Sayre Lawrence M LM

The Journal of organic chemistry 20090101 1

We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (K(m) 183 +/- 14 nM) and yet is metabolized fairly quickly (k(cat) 0.690 +/- 0.010 s(-1)), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorom ...[more]

PMID: 19053593

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Project description:Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. βAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). βAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

Dataset Information

Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.

Publications

Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.

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