title: Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay.
abstract: The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.
authors.affiliation: Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, NJ 08901, United States.
authors.forename: Azam
authors.initials: A
authors.lastname: Hassaninasab
authors.affiliation: Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, NJ 08901, United States.
authors.forename: Gil-Soo
authors.initials: GS
authors.lastname: Han
authors.affiliation: Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, NJ 08901, United States. Electronic address: gcarman@rutgers.edu.
authors.forename: George M
authors.initials: GM
authors.lastname: Carman
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008071:Lipoprotein Lipase / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D017830:Octoxynol / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D010712:Phosphatidic Acids / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005994:Glycerophosphates; D010078:Oxazines; D010712:Phosphatidic Acids; C014180:resorufin; D017830:Octoxynol; D006861:Hydrogen Peroxide; D005993:Glycerolphosphate Dehydrogenase; C098831:glycerol-3-phosphate oxidase; D008071:Lipoprotein Lipase
keywords: Coupled enzyme assay; Fluorometric assay; Phosphatidic acid; Phosphatidic acid phosphatase; Yeast