In vitro characterization of membrane associated phospholipase C from normal and Kirsten sarcoma virus-transformed NIH 3T3 cells
Transformation of NIH 3T3 cells with Kirsten sarcoma virus (Ki-SV) increased phosphatidylinositol (PI) metabolism. This suggests possible alterations in the phospholipase C (PLC) and PIP/sub 2/-phosphodiesterase (PIP/sub 2/-PDE) activities responsible for hydrolysis of PI and PIP/sub 2/ with Ki-SV transformation. An in vitro assay is employed to study the hydrolysis of exogenously added (/sup 3/H)PI and (/sup 3/H)PIP/sub 2/ with membranes prepared from normal and Ki-SV transformed cells. Association of these activities with membranes appears to be differentially mediated by metals (Ca/sup 2 +/) since chelator treatment dissociates PLC from the particulate fraction. Hydrolysis of PIP/sub 2/ is markedly enhanced (10 fold) by introducing (/sup 3/H)PIP/sub 2/ to membrane preparations in vesicles prepared with excess phosphatidylethanolamine. These activities are dependent on Ca/sup 2 +/ and exhibit a progressive increase in activity between 10/sup -7/M and 10/sup -3/M Ca/sup 2 +/. The optimal pH for PIP/sub 2/-PDE is 7.0, whereas PI specific PLC exhibits optimal activity at pH 5.5. With this in vitro assay system it is possible to demonstrate that GTP-..gamma..-S addition to isolated membranes stimulates PIP/sub 2/-PDE to hydrolyze exogenously added (/sup 3/H)PIP/sub 2/. This should allow direct studies to determine possible differences in GTP-dependent regulation of PI and PIP/sub 2/ hydrolysis with membranes prepared from normal and transformed cells.
- Research Organization:
- National Institute of Health, Bethesda, MD
- OSTI ID:
- 5328012
- Report Number(s):
- CONF-8606151-; TRN: 86-031552
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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