Study of the mechanism of phospholipase C activation via a new GTP binding protein

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This study was undertaken to elucidate the GTP binding protein to activate phospholipase C (PLase C) which is not a substarate for pertussis toxin (IAP) in human astrocytoma cells. The stimulation of TXA_2-receptors as well as muscarinic, H_1-histamine and bradykinin resulted in activation of PLase C in an IAP insensitive manner. The GTP binding protein involved in these agonists-induced PLase C activations is not a substrate for IAP. Pretreatment of cells with agonists elicited the reduction of GTPgammaS-induced accumulation of inositol phosphates (IP) in membrane preparations, reflecting from a functional reduction of signal transduction after agonist treatment of intact cells. Heavy peak fraction of membranes after sucrose gradient separation contained receptors and GTP binding proteins. Receptors together with GTP binding proteins moved from heavy membrane fraction to light peak fraction after treatment of the intact cells with agonists. The reduction of GTP binding protein in heav … More y peak fraction after agonist treatment was analyzed by a photoaffinity labelling with [^<35>S]GTPgammaS. The 32 kDa GTP binding protein was reduced in Heavy peak fraction after treatments of the intact cells with agonists. The 32 kDa GTP binding protein might be involved in receptor-mediated activation of phospholipase C. Then, the 32 kDa GTP binding protein was purified from the porcine brain membranes. There is a 32 kDa GTP binding protein in the membranes, determined by a photoaffinity labelling with [^<32>P]alpha-GTP. The 32 kDa GTP binding protein was solubilized with 1 % lubrol from cholate-inextracted membranes. The 32 kDa gtp binding protein might be hydrophobic, and was not ADP-ribosylated with IAP. The 32 kDa GTP binding protein was purified by column chromatographies of DEAE-sephacel, sephacryl S-200 and hydroxyapatite. Although the 32 kda protein corresponded to [^<35>S]GTPgammaS binding activities was appeared in hydroxyapatite column chromatography, further purifications were necessary. In conclusion, there is the 32 kDa GTP binding protein in brain membranes as well as astrocytoma cells which is a candidate for a GTP binding pritein to activate phospholipase C in an IAP-insenitive manner. Less