|Budget Amount *help
¥7,200,000 (Direct Cost : ¥7,200,000)
Fiscal Year 1995 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1994 : ¥5,600,000 (Direct Cost : ¥5,600,000)
[I] Activation mechanism of phospholipase D : A low mocelcular weight GTP-binding protein (small G protein), ADP-ribosylation factor (ARF), has recently been identified as a phospholipase D (PLD) activator. We found that the calcium-binding protein, Calmodulin (CaM), also sctivates PLD in the streptolysin O-permeabilized, cytosol-depleted rabbit peritoneal neutrophils. Furthermore, it was found that CaM and ARF synergistically activate PLD when they are simulteneously reconstituted with the permeabilized neutrophils.
We also demonstrated that RhoA,which is a member of small G protein superfamily and specificaly ADP-ribosylated by Clostridium botulinum C3 exoenzyme, activates the ARF-sensitive PLD partially purified rat brain membrane fraction. RhoA and ARF again synergistically activated the PLD.Furthermore, it was suggested that the post-translational modification of RhoA (gelanylgelanylation) is required for the ability of RhoA to activate the PLD.
In addition to the protein activators of PLD described above, phosphatidylinositol 4,5-bisphosphate (PIP_2) is identified as a cofactor for the small G protein activation of PLD in vitro. Several lines of evidence suggested that PLD translocates to phospholipid vesicles containing PIP_2 and the PLD substrate (phosphatidylcholine), due to the specific interaction with PIP_2, then is activated by the small G proteins on the vesicles, thereby hydrolyzing phosphatidylcholine.
[II] Purification of phospholipase D and its cDNA cloning : We are trying to highly purify PLD from rat or rabbit brain membrane fraction. At present, however, PLD is partially purified from rat brain membranes.
Recently, cDNA of the ARF-sensitive PLD has been cloned from HeLa cells by Hommand et al. We are investigating whether or not there exist PLD isozymes by using cDNA of the ARF-sensitive PLD as a probe.