A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity.
Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression.
Ahmad
Syed
S
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
Carter
Jade J
JJ
Scott
John E
JE
D043303:Aryldialkylphosphatase / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006801:Humans; D006912:Hydroxyquinolines / Q000737:chemistry / Q000494:pharmacology; D010755:Organophosphates / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014468:Umbelliferones / Q000737:chemistry / Q000494:pharmacology
C492761:7-diethylphospho-6,8-difluoro-4-methylumbelliferyl; D005456:Fluorescent Dyes; D006912:Hydroxyquinolines; D010755:Organophosphates; D014468:Umbelliferones; C107724:2-hydroxyquinoline; D043303:Aryldialkylphosphatase