Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine

The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the t ransfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar V-max values, sug gesting that the formation of the phosphatidyl-enzyme intermediate is the r ate-limiting step. The enzyme had an absolute requirement for Ca2+, and the optimum concentration was approximately 40 mM CaCl2. K-Ca(app) was calcula ted to be 5.6 +/- 0.74 mM for the hydrolytic and 10 +/- 0.97 mM for the tra nsphosphatidylation reaction. Both activities reached a maximum at pH 5.5, independent of Ca2+ concentration. Kinetic studies showed that the Km(app) for the glycerol in the transphosphatidylation reaction is 388 +/- 37 mM. K m(app) for the lysophosphatidylcholine depended on Ca2+ concentration and f ell between 1 and 3 mM at CaCl2 concentrations from 4 to 40 mM. SDS, TX-100 , and CTAB did not activate the enzyme as reported for phosphatidylcholine hydrolysis; on the contrary, reaction rates decreased at detergent concentr ations at or above that of lysophosphalidylcholine. (C) 2000 Elsevier Scien ce Ireland Ltd. All rights reserved.