The Physiological Roles of G-Protein and Phospholipase C in Neoplastic Cells

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1. Phospholipase of mastocytoma : Two forms of phosphoinositide-specific phospholipase C (PLCI and PLCII) were purified from the cytosolic fraction of mastocytoma P-815 cells. Both enzymes had high specificities for PIP_2 ; especially, PLCI showed approximately 100times higher activity with PIP_2 as substrate than that with PI at 10^<-7> M Ca^<2+> which seems to be physiological concentration in the cells. These results suggest that both PLC have important role for the signal transduction through Ca^<2+> mobilization.2. A soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) in mastocytoma : 65% of total PT-substrate in P-815 cell homogenate was found to be detected in the cytosolic fraction of the cells. The Mr or the cytosolic PT-substrate was estimated to be 100kDa on an Ultogel AcA44 column. Although the cytosolic PT-substrate was identified as Gi_<2alpha>, it did not contain betagamma complex.3. Translocation of the Gi_<2alpha> from the membrane to the cytosol : Incubation of the P-815 membrane with GTPgammaS caused a small release (10%) of Gi_<2alpha> from the membrane. Although cytosol alone had no ability to release the Gi_<2alpha> from the membrane, it markedly augmented the release induced by GTPgammaS, about 50% of the total Gi_<2alpha> being released by 30 min. The GTPgammaS-induced release and its enhancement by the cytosol were specific for GTP and GTPgammaS. The Mr of the Gi_<2alpha> released by the cytosol plus GTPgammaS from the membrane was estimated to be 100kDa on an Ultrogel AcA44 column. In contrast, the Gi_<2alpha> released by GTPgammaS alone had Mr of 40kDa, but it was eluted at the position of Mr=100kDa when it was incubated with the cytosol.These results demonstrate that cytosol contain some factor (s) that promotes the release of GTP-activated Gi_<2alpha> from the membrane and that the released Gi_<2alpha> exists in the cytosol as a soluble complex with unidentified component (s) in mastocytoma cells.