title: An octaethylene glycol monododecyl ether-based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin.
abstract: Patatin was extracted from potato tubers (Solanum tuberosum L. cv. Spunta) and purified to homogeneity by ammonium sulfate salt fractionation and one sole chromatographic step. A spectrophotometric mixed micellar assay for patatin lipid acyl hydrolase (LAH) activity was designed with the detergent octaethylene glycol monododecyl ether (C12E8). Patatin LAH used p-nitrophenyl butyrate (PNP-butyrate) as substrate when solubilized in (C12E8) micelles. In the mixed micellar system, patatin LAH responds to the PNP-butyrate surface concentration expressed as mol% (= [PNP-butyratel x 100/([detergentl critical micellar concentration)) and not to the molarity of PNP-butyrate. The kinetic parameters were determined; Vmax was independent of the mixed micelle concentration, as was Km, when expressed as mol%. However, Km was dependent on C12E8 concentration when expressed in molar concentration. C12E8/PNP-butyrate proved to be a reliable system for assaying patatin LAH activity and is superior to the commonly used Triton X-100 and SDS methods. It permits investigation of the substrate requirements of patatin LAH activity because the concentration-independent Km can be determined both in mol% and as the absolute number of substrate molecules per micelle. In addition, the detergent did not affect the enzyme activity.
authors.affiliation: Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Spain. mja@um.es
authors.forename: M
authors.initials: M
authors.lastname: Jiménez
authors.forename: J
authors.initials: J
authors.lastname: Escribano
authors.forename: M
authors.initials: M
authors.lastname: Pérez-Gilabert
authors.forename: S
authors.initials: S
authors.lastname: Chazarra
authors.forename: J
authors.initials: J
authors.lastname: Cabanes
authors.forename: F
authors.initials: F
authors.lastname: García-Carmona
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008823:Micelles; D010940:Plant Proteins / Q000378:metabolism*; D011092:Polyethylene Glycols; D011198:Solanum tuberosum / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D003902:Detergents; D008823:Micelles; D010940:Plant Proteins; C043222:patatin protein, Solanum tuberosum; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D002265:Carboxylic Ester Hydrolases