Augmentation by calmodulin of ADP-ribosylation factor-stimulated phospholipase D activity in permeabilized rabbit peritoneal neutrophils.
Activation of the membrane-bound phospholipase D (PLD) requires cytosolic factor(s), and ADP-ribosylation factor (ARF) has been identified as a cytosolic PLD activator. In the present study, we demonstrate that calmodulin (CaM) and ARF are both involved in PLD activation in rabbit peritoneal neutrophils. The PLD activity of streptolysin O-permeabilized, cytosol-depleted rabbit neutrophils was significantly enhanced when the permeabilized cells were reconstituted with bovine brain cytosol in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas there was little activation of the enzyme in the absence of cytosol. The GTP gamma S-stimulated PLD activity in the presence of cytosol was augmented on increasing the concentration of free Ca2+. The PLD activity stimulated by GTP gamma S and Ca2+ in this system was inhibited by the calmodulin inhibitor W-7. These findings suggest that CaM plays a role as a cytosolic PLD activator. Moreover, highly purified CaM alone, as well as partially purified ARF alone, promoted a slight stimulation of the PLD activity in permeabilized neutrophils. Interestingly, ARF-stimulated PLD activity was augmented by CaM in the presence of GTP gamma S and Ca2+. This augmentation was again inhibited by W-7, as well as by the structurally unrelated CaM inhibitor trifluoperazine. These data imply that CaM stimulates the PLD activity of rabbit neutrophils in concert with ARF.
Takahashi
K
K
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Tago
K
K
Okano
H
H
Ohya
Y
Y
Katada
T
T
Kanaho
Y
Y
D020727:ADP-Ribosylation Factors; D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology; D002147:Calmodulin / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D010739:Phospholipase D / Q000096:biosynthesis / Q000187:drug effects*; D011817:Rabbits
D002147:Calmodulin; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors