Isolation and characterization of a 66-kDa protein from rat liver plasma membrane with RhoA-stimulated phospholipase D activity.
A 66-kDa molecular weight protein with phospholipase D activity was solubilized and partially purified from rat liver plasma membrane. The activity and regulation of this phospholipase D have been characterized. Immunoblot analyses indicated that the enzyme was distinct from hPLD1 and PLD2, but was recognized by an antibody to the 12 terminal amino acids of PLD1. PLD activity was stimulated by 1-100 microM Ca(2+) and Mg(2+) and displayed a pH optimum of 7.5. Activity was inhibited by both saturated and unsaturated fatty acids. This PLD was activated in an ATP-independent manner by the PKC isozymes alpha and betaII but not activated by other PKC isozymes. It was also stimulated by the small G-proteins RhoA and ARF. RhoA stimulated the greatest activation, followed by ARF and PKC(alpha). This enzyme was further activated in a synergistic manner when combinations of PKC(alpha) and RhoA or ARF were used. This enzyme displayed a greater response activation by RhoA than to activation by ARF. While a potential breakdown product of PLD1, activation by RhoA indicates that the PLD characterized here is distinct from the other PLDs cloned or isolated to date.
Dunkirk
Shawn G
SG
Department of Chemistry and Department of Biology, Minnesota State University Moorhead, Moorhead, Minnesota 56563, USA.
Wallert
Mark A
MA
Baumgartner
Matt L
ML
Provost
Joseph J
JJ
D020727:ADP-Ribosylation Factors / Q000502:physiology; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology*; D002845:Chromatography; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D010739:Phospholipase D / Q000302:isolation & purification* / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D016622:Silver Staining; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D011506:Proteins; D011493:Protein Kinase C; D010739:Phospholipase D; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein