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 transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar V(max) values, suggesting that the formation of the phosphatidyl-enzyme intermediate is the rate-limiting step. The enzyme had an absolute requirement for Ca²⁺, and the optimum concentration was approximately 40 mM CaCl₂. K(Ca)(app) was calculated to be 8.6±0.74 mM for the hydrolytic and 10±0.97 mM for the transphosphatidylation 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. Km(app) for the lysophosphatidylcholine depended on Ca²⁺ concentration and fell 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 concentrations at or above that of lysophosphatidylcholine. Copyright (C) 2000 Elsevier Science Ireland Ltd.