Abstract
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.
Highlights
¶ To whom correspondence should be addressed: Dept. of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425
Effect of Membrane Extracts on [35S]GTP␥S Binding to Bovine Brain G-protein—A solution phase assay was developed to search for substances that influenced nucleotide binding to heterotrimeric G-protein
Membrane extracts were incubated with bovine brain G-protein in the presence of GDP, and aliquots were added to binding tubes containing [35S]GTP␥S to determine effects of extracts on nucleotide binding to G-protein
Summary
Tissue culture supplies were obtained from JRH Bioscience (Lenexa, KS). Acrylamide, bisacrylamide, and SDS were purchased from Bio-Rad. Nitrocellulose and polyvinylidene difluoride membranes were obtained from Schleicher & Schuell. Antisera recognizing Go and caveolin (common for caveolin-1, -2, and -3) were kindly provided by Dr Eva J. Neer (Harvard Medical School) [25] and Dr Michael P. Lisanti (Whitehead Institute for Biomedical Research) [23, 24], respectively. The monoclonal antibody to ␣ and  tubulin were obtained from Amersham. Gel filtration molecular weight markers were purchased from Sigma and Boehringer Mannheim. Ecoscint A was purchased from National Diagnostics (Manville, NJ). Guanosine triphosphate, Thesit (polyoxyethylene-9-lauryl ether), monoclonal antibodies to neuromodulin (clone 91E12), and -amyloid precursor protein (clone 22C11) were obtained from Boehringer Mannheim
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