Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta.
In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP2 and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 microM GTP gamma S (6-14-fold) and by the small G proteins 1 microM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human placenta, which is stimulated by PIP2, but not by oleate (Vinggaard, A. M. & Hansen, H. S. (1995) Biochim. Biophys. Acta 1258, 169-176). Here we show that oleic acid and alpha-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important for the inhibitory effect. Furthermore, placental membrane PLD was activated by 30 microM GTP gamma S (4-fold) and by mArf3 (1 microM) and RhoA (0.37 nM) by a factor of 3 and 2, respectively. The solubilized membrane phospholipase D was partially purified to a basal specific activity of 25-37 nmol/min/mg. This preparation was devoid of endogenous RhoA and Arf and could not be stimulated by GTP gamma S. However, mArf3 (1 microM) still activated this partially purified membrane PLD, whereas RhoA (0.37 nM) was not able to activate this PLD fraction. In conclusion, our results suggest that the human placenta contains a PLD that is located both in the cytosol and the membranes, and that is activated by PIP2, mArf3 and RhoA but inhibited by oleate.
Vinggaard
A M
AM
Department of Biological Sciences, Royal Danish School of Pharmacy, Copenhagen, Denmark.
Provost
J J
JJ
Exton
J H
JH
Hansen
H S
HS
D020727:ADP-Ribosylation Factors; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019301:Oleic Acid / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010739:Phospholipase D / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D015398:Signal Transduction / Q000502:physiology; D017962:alpha-Linolenic Acid / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D019269:Phosphatidylinositol 4,5-Diphosphate; D017962:alpha-Linolenic Acid; D019301:Oleic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein