In vitro characterization of membrane associated phospholipase C from normal and Kirsten sarcoma virus-transformed NIH 3T3 cells

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5328012

Talwar, H S; Thomas, T P; Bassin, R; Anderson, W B

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-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550901* -- Pathology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ANIMAL CELLS
CALCIUM COMPOUNDS
CARBOHYDRATES
CARBOXYLESTERASES
CELL CONSTITUENTS
CELL MEMBRANES
CELL TRANSFORMATIONS
CHEMICAL REACTIONS
CONNECTIVE TISSUE CELLS
DECOMPOSITION
DISEASES
ENZYME ACTIVITY
ENZYMES
ESTERASES
ESTERS
FIBROBLASTS
HYDROLASES
HYDROLYSIS
IN VITRO
INOSITOL
INOSITOLS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPASE
LIPIDS
LYSIS
MEMBRANES
METABOLISM
MICROORGANISMS
MONOSACCHARIDES
NEOPLASMS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PARASITES
PHOSPHOLIPIDS
SACCHARIDES
SARCOMAS
SOLVOLYSIS
SOMATIC CELLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VIRUSES