Enzymatic determination of phospholipase D activity with choline oxidase

• Published in: Journal of biochemistry (Tokyo) Vol. 83; no. 3; p. 677
• Main Authors: Imamura, S, Horiuti, Y
• Format: Journal Article
• Language: English
• Published: England 01-03-1978
• Subjects: Alcohol Oxidoreductases - metabolism; Aldehyde Oxidoreductases - metabolism; Arthrobacter - enzymology; Betaine; Brassica - enzymology; Choline - analysis; Methods; Phospholipases - analysis
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a131960

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.