An octaethylene glycol monododecyl ether-based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin.
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.
Jiménez
M
M
Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Spain. mja@um.es
Escribano
J
J
Pérez-Gilabert
M
M
Chazarra
S
S
Cabanes
J
J
García-Carmona
F
F
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
D003902:Detergents; D008823:Micelles; D010940:Plant Proteins; C043222:patatin protein, Solanum tuberosum; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D002265:Carboxylic Ester Hydrolases