The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein.
Journal of Biological ChemistryPublished 1 June 1986Open access
Richard Kahn, Alfred G. Gilman
Citations534
SJR quartileQ1
SJR score1.71
SNIP1.00
Generate an AI Snapshot to get a quick, structured summary of this paper.
Study Snapshot
ObjectiveStudy objective
MethodsResearch methodology
PopulationPopulation studied
Sample sizeSample sizes
OutcomesStudy outcomes here
ResultsStudy results comes here
LimitationsResearch study limitations comes here
A concise AI-generated summary of the paper will appear here once you click Generate AI Snapshot.
TL;DR
Improved methods for the purification of ARF from bovine brain are described, and although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected.
Abstract
A membrane-bound protein cofactor (ARF) is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory regulatory component (Gs) of adenylate cyclase. Improved methods for the purification of ARF from bovine brain are described. ARF has a high-affinity binding site for guanine nucleotides. Binding of GTP or GTP gamma S to ARF is necessary for the activity of the cofactor; GDP X ARF does not support ADP-ribosylation of Gs. Although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected. Cholera toxin-dependent activation of adenylate cyclase thus requires two guanine nucleotide binding proteins.
Keywords
Biochemistry, Genetics and Molecular Biology
Analytical BiochemistryA rapid, sensitive, and specific method for the determination of protein in dilute solution
2,524 Citations1973W. Schaffner, Charles Weissmann
This protein assay is described in which the sample is precipitated with trichloroacetic acid in the presence of sodium dodecylsulfate, filtered off on a Millipore membrane and stained with Amidoschwarz 10B, and its absorbance determined at 630 nm.
CellG proteins and dual control of adenylate cyclase
1,813 Citations1984Alfred G. Gilman
It is clear that detailed understanding of the mechanism of regulation of CAMP synthesis will soon be achieved from study of the interactions of purified components that have been reconstituted in lipid bilayers of defined composition.
Journal of Biological ChemistryIsolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain.
1,309 Citations1984Paul C. Sternweis, Janet D. Robishaw
The alpha subunit of GO was isolated without the use of ligands known to dissociate other G proteins, and should be of great utility in elucidating the mechanism of action of this family of GTP-binding proteins.
CellIn yeast, RAS proteins are controlling elements of adenylate cyclase
1,143 Citations1985Takashi Toda, Isao Uno +8 more
S. cerevisiae strains containing RAS2val19, a Ras2 gene with a missense mutation analogous to one that activates the transforming potential of mammalian ras genes, have growth and biochemical properties strikingly similar to yeast strains carrying IAC or bcy1.
Journal of VirologyMonoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family
820 Citations1982Mark E. Furth, Lenora J. Davis +2 more
Eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus are isolated, suggesting that an endogenous gene closely related to Kirsten-ras is expressed in these cells.
Proceedings of the National Academy of SciencesIntrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules.
616 Citations1984W. Wayt Gibbs, Irving S. Sigal +2 more
The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex.
Biochimica et Biophysica Acta (BBA) - Reviews on BioenergeticsThe role of guanosine 5′-triphosphate in polypeptide chain elongation
560 Citations1978Yoshito Kaziro
Journal of Biological ChemistryThe guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding.
549 Citations1982John K. Northup, M D Smigel +1 more
There is no reversal of activation or binding under activating conditions and the rate constant for activation is unchanged from 10 mi to 100 PM GTPyS, so reversal of binding and activation, which occurs only in the absence of divalent cation, is not a first order process.
Journal of Biological ChemistryPurification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin.
410 Citations1984Richard Kahn, Alfred G. Gilman
The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.
Annual Review of BiochemistryActivation of Adenylate Cyclase by Choleragen
394 Citations1979Joel Moss, M Vaughan
An attempt is made to evaluate the mechanism of action of NAD Glycohydrolase and ADP-Ribosyltransferase on GTP-Binding Protein and GTPase Activity in response to the presence of Gangliosides and Their Oligosaccharides in Choleragen.
Journal of Biological ChemistryThe role of iron in the activation-inactivation of aconitase.
380 Citations1983M C Kennedy, M H Emptage +2 more
It is concluded that, in analogy to cluster loss during activation in absence of added iron, the appearance of 59Fe in more than one cluster site can be due to complete breakdown and rebuilding of clusters, and exchange into intact clusters cannot be ruled out.
BiochemistryReconstitution of catecholamine-stimulated guanosine triphosphatase activity
212 Citations1983Douglas R. Brandt, Tomiko Asano +2 more
Beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver to hydrolyze GTP in response to beta-adrenergic agonists.
NatureThe ras oncogene product p21 is not a regulatory component of adenylate cyclase
209 Citations1985Suzanne K. Beckner, Seisuke Hattori +1 more
The structural and functional similarities between p21 and Ns and Ni are examined and it is found no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.
Journal of Biological ChemistryADP-ribosylation of Gs promotes the dissociation of its alpha and beta subunits.
196 Citations1984Richard Kahn, Alfred G. Gilman
This work has utilized purified reactants and cofactors to examine the form of the stimulatory guanine nucleotide-binding regulatory component of adenylate cyclase that serves as a substrate for ADP-ribosylation by cholera toxin and investigated some of the consequences of that covalent modification.
Proceedings of the National Academy of SciencesReconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes.
177 Citations1977Elliott M. Ross, Alfred G. Gilman
The uncoupled and adenylate cyclase-deficient variants of the S49 cell are not complementary and the effects of beta-adrenergic agonists and antagonists on the reconstituted system reproduce those that are characteristic of the wild-type S49 lymphoma cell.
Journal of Biological ChemistryRequirements for cholera toxin-dependent ADP-ribosylation of the purified regulatory component of adenylate cyclase.
173 Citations1982L. S. Schleifer, Richard Kahn +4 more
The initial rate of activation of the regulatory component of adenylate cyclase by toxin was found to be linearly related to the amount of factor present in the reaction, which has allowed the quantitation and partial purification of the factor from turkey erythrocyte membranes.
Journal of Supramolecular StructureRequirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin
63 Citations1979Keiichi Enomoto, D. Michael Gill
The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates, but after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP.
Proceedings of the National Academy of SciencesCholeragen activation of solubilized adenylate cyclase: Requirement for GTP and protein activator for demonstration of enzymatic activity
63 Citations1977Joel Moss, Martha Vaughan
It appears that the cyclase system is dissociable into several components, all of which may be necessary for optimal regulation of activity, and one of these is the heat-stable calcium-dependent protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase.
Journal of VirologyPhotoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli
57 Citations1984Robert B. Stein, Pierre Robinson +1 more
The expression in Escherichia coli of v-rasH is reported to produce a biochemically active p21 fusion protein which retains both guanine nucleotide binding and autophosphorylating activity.
BiochemistryEffects of nucleoside triphosphates on choleragen-activated brain adenylate cyclase
44 Citations1980Seishi Nakaya, Joel Moss +1 more
It appears that GTP, which was effective in micromolar concentrations, plays an important role not only in the activation of adenylate cyclase by choleragen but also in the stabilization and expression of the catalytic function of the activated enzyme.