TY - JOUR

T1 - Characterization and partial purification of phospholipase D from human placenta

AU - Vinggaard, Anne Marie

AU - Hansen, Harald S.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - 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 K(m) of 33 mol% (or 0.8 mM). Ca and Mg 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 (EC = 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) µM) and (±) propranolol (53 µM) had no direct effect on PLD activity. The present results form the basis for further purification of a PLD from human tissue.

AB - 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 K(m) of 33 mol% (or 0.8 mM). Ca and Mg 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 (EC = 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) µM) and (±) propranolol (53 µM) had no direct effect on PLD activity. The present results form the basis for further purification of a PLD from human tissue.

UR - http://www.scopus.com/inward/record.url?scp=0029160103&partnerID=8YFLogxK

U2 - 10.1016/0005-2760(95)00121-R

DO - 10.1016/0005-2760(95)00121-R

M3 - Journal article

AN - SCOPUS:0029160103

VL - 1258

SP - 169

EP - 176

JO - Biochimica et Biophysica Acta - Lipids and Lipid Metabolism

JF - Biochimica et Biophysica Acta - Lipids and Lipid Metabolism

SN - 0005-2760

IS - 2

ER -