|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1993 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1992 : ¥1,400,000 (Direct Cost : ¥1,400,000)
It has recetly been reported that, in vaious types of mammalian cells, many agonists activates phospholipase D thrugh receptors on plasma membranes. The mechanism f the enzyme activation is nt yet been clarified. To investigate the activation mechanism f phospholipase D in rabbit peritoneal neutrophls, permeabilized neutrophils and cytosol prepared from rat brain were reconstituted and the phoshlipase D activation was investigated under various conditions.
[I] Phosholipase D ctivation in reconstituted system containing permeabilized neutrohils and cytosol - After prelabelng 1-omicron-alkyl-phoshatidylcholine in rabbit peritoneal neutrophils with [^3H]lyso platelet-activating factor at 37ﾟC for 2 hr, the cells were permeabilized with 1 U/ml of streptolysin Oat 37ﾟC for 20 mn to remove cytosol. The permeabilized cells were incubated in the presence of 1% ethanol under vaius conditions, then [^3H]phsphatidylethanl (PEt), an unambiguous product of phospholipase D, were determined. When cyto
sol prepared from rat brain were reconstituted with permeabilized cells, GTPgammaS/Ca^<2+>/Mg-ATP significantly stimulted [^3H]PEt formation. The stimulation was not observed in the absence of cytosol. Since phospholipase D locates on plasma membranes, these results suggest that phospholipase D activation requires cytosolic factor(s).
[II] Phospholipase D activation factor : Since it is likely that a Ca^<2+>-dependent factor is, at least in part, involved in phospholipase D activation in rabbit peritoneal neutrophils it was investigated whether or not calmodulin (CaM) is involved in phospholipase D activation in the reconstituted system described above. CaM-free cytosol was prepared by flufenazine-immobilized affinity column chromatography. CaM bound to the column as eluted with EGTA slution. In permeabilized neutrophils reconstituted with CaM-free cytosol, GTPgammaS/Ca^<2+>/Mg-ATP stimulated phospholipase D to much less extent than in the reconstitute dsystem with cytosol. When CaM euted frm the column was added to the reconstituted system with CaM-free cytosol, phospholipase D activation by GTPgammaS/Ca^<2+>/Mg-ATP was maredly augmented. The urified CaM from bovine brain cytosl had the same effect with CaM eluted from the affinity column. From these results, it is suggested that CaM ia, at least in part, involved in phospholipase D activation as a cytosolic factor in rabbit peritoneal neutrophils.