CHARACTERIZATION AND PARTIAL-PURIFICATION OF PHOSPHOLIPASE-D FROM HUMAN PLACENTA

We report the existence in the human placenta of a phosphatidylcholine -hydrolyzing phospholipase D (PLD) activity, which has been characteri zed and partially purified. Triton X-100 effectively solubilized PLD f rom the particulate fraction of human placenta in a dose-dependent man ner. However, Triton X-100 caused decreasing enzyme activities. Maximu m transphosphatidylation was obtained with 2% ethanol. The enzyme was found to have a pH optimum of 7.0-7.5 and an apparent K-m of 33 mol% ( or 0.8 mM). Ca2+ and Mg2+ was not required for the enzyme activity. Ad dition of phosphatidyl-4,5-bisphosphate, but not phosphatidylethanolam ine, to the substrate mixture gave rise to a pronounced dose-dependent increase in PLD activity (EC(50) = 0.3 mol%), suggesting a regulatory role of this phospholipid in PLD action. The enzyme was inhibited by sodium oleate when partly or fully substituting for octylglucoside in the substrate mixture. The PLD activity was enriched 15-fold by solubi lization and purification on a DEAE-Sepharose column. N-Ethylmaleimide (10 mM) markedly inhibited the purified enzyme, indicating the presen ce of free thiol groups on PLD. Sphingosine (20 mu M) and (+/-) propra nolol (53 mu M) had no direct effect on PLD activity. The present resu lts form the basis for further purification of a PLD from human tissue .