Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay.
Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl] PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (Vm = 9.8 x 10(-3) micromol/min x microg protein, Km = 7.8 microM, Vm/Km = 1.3 min(-1) x microg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.
Jiménez-Atiénzar
M
M
Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Espinardo 30071, Murcia, Spain. mja@um.es
Cabanes
J
J
Gandía-Herrero
F
F
Escribano
J
J
García-Carmona
F
F
Pérez-Gilabert
M
M
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011198:Solanum tuberosum / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods*
D010713:Phosphatidylcholines; D010743:Phospholipids; D010940:Plant Proteins; C043222:patatin protein, Solanum tuberosum; D003840:Deoxycholic Acid; C002448:1,2-linoleoylphosphatidylcholine; D008084:Lipoxygenase; D002265:Carboxylic Ester Hydrolases; D010741:Phospholipases A