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 characterized and partially purified. Triton X-100 effectively solubilized PLD from the particulate fraction of human placenta in a dose-dependent manner. However, Triton X-100 caused decreasing enzyme activities. Maximum transphosphatidylation was obtained with 2% ethanol. The enzyme was found to have a pH optimum of 7.0-7.5 and an apparent Km of 33 mol% (or 0.8 mM). Ca2+ and Mg2+ was not required for the enzyme activity. Addition of phosphatidyl-4,5-bisphosphate, but not phosphatidylethanolamine, to the substrate mixture gave rise to a pronounced dose-dependent increase in PLD activity (EC50 = 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 solubilization and purification on a DEAE-Sepharose column. N-Ethylmaleimide (10 mM) markedly inhibited the purified enzyme, indicating the presence of free thiol groups on PLD. Sphingosine (20 microM) and (+/-) propranolol (53 microM) had no direct effect on PLD activity. The present results form the basis for further purification of a PLD from human tissue.
Vinggaard
A M
AM
Department of Biological Sciences, Royal Danish School of Pharmacy, Copenhagen.
Hansen
H S
HS
D002451:Cell Compartmentation; D000431:Ethanol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007477:Ions; D050356:Lipid Metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010739:Phospholipase D / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D012995:Solubility; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D007477:Ions; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D013439:Sulfhydryl Reagents; D000431:Ethanol; D010739:Phospholipase D; D005033:Ethylmaleimide