Enzymatic determination of phospholipase D activity with choline oxidase.
A new enzymatic method was developed for the assay of phospholipase D [phosphatidylcholine phosphatidohydrolase EC 3.1.4.4] from cabbage leaves using choline oxidase from Arthrobacter globiformis cells. The method was based on the estimation of choline by the following series of enzymatic reactions after ending the phospholipase D reaction: Choline + 202 + h2o Choline oxidase Betaine + 2H2O2 2H2O2 + Phenol + 4-Aminoantipyrine Peroxidase Quinoneimine dye + 4H2O The amount of choline was proportional to the amount of resulting quinoneimine dye with an absorbance maximum at 500 nm. The phospholipase D reaction (choline liberation) was carried out at pH 5.5 in the presence of Ca2+ ions and ended by adding EDTA in conc. Tris-HCl buffer, pH 8, to give a final pH of around 8. The initial rate of the phospholipase D reaction was proportional to the enzyme concentration over the absorbance change range of 0 to 0.25 (equivalent to 0-21 micron of choline) under the optimal reaction conditions.
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology; D001622:Betaine; D001937:Brassica / Q000201:enzymology; D002794:Choline / Q000032:analysis; D008722:Methods; D010740:Phospholipases / Q000032:analysis*
D001622:Betaine; D000429:Alcohol Oxidoreductases; D000445:Aldehyde Oxidoreductases; D010740:Phospholipases; D002794:Choline