Characterization of a G-protein activator in the neuroblastoma-glioma cell hybrid NG108-15.
Purified bovine brain G-protein was used in a solution phase assay to identify membrane-associated proteins that influenced the activation of heterotrimeric G-proteins. Detergent-solubilized membrane extracts from the neuroblastoma-glioma cell hybrid NG108-15, but not the parent C6B4 glioma cell line, increased [35S]GTPgammaS binding to purified G-protein by approximately 460%. The G-protein activator was heat-sensitive, and the magnitude of its action was related to the amount of extract protein. The biophysical and biochemical properties of the G-protein activator were determined using DEAE ion exchange chromatography, gel filtration, and a lectin affinity matrix. In the presence of added GDP (1 microM), the enriched G-protein activator increased the initial rate of [35S]GTPgammaS binding to brain G-protein by up to 4-fold. In the absence of added GDP, the G-protein activator elicited an initial burst in [35S]GTPgammaS binding to brain G-protein within the first 30 s, after which the rate of nucleotide binding to G-protein was similar in the absence or presence of the G-protein activator. The stimulation of nucleotide binding to brain G-protein by the activator was also observed after resolution of Galpha from Gbetagamma. The G-protein activator was distinct from other proteins (neuromodulin, tubulin, and beta-amyloid precursor protein) that influence nucleotide binding to G-protein, indicating the existence of a novel signal accelerator.
Sato
M
M
Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Ribas
C
C
Hildebrandt
J D
JD
Lanier
S M
SM
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006822:Hybrid Cells; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins