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 Ca(2+), and the optimum concentration was approximately 40 mM CaCl(2). 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 Ca(2+) 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 Ca(2+) concentration and fell between 1 and 3 mM at CaCl(2) 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.
Virto
C
C
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100, Lund, Sweden. carmen.virto@biotek.lu.se
Svensson
I
I
Adlercreutz
P
P
D001937:Brassica / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism*; D008823:Micelles; D010739:Phospholipase D / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D003902:Detergents; D008244:Lysophosphatidylcholines; D008823:Micelles; D010739:Phospholipase D; D002118:Calcium