Regulation by guanosine 3':5'-cyclic monophosphate of phospholipid methylation during chemotaxis in Dictyostelium discoideum (original) (raw)
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Proceedings of the National Academy of Sciences, 1979
Suspensions of cyclic AMP sensitive cells of Dictyostelium discoideum responded to a cyclic AMP pulse with increased methylation of a protein of molecular weight about 120,000 and increased phospholipid demethylation. Protein methylation reached its peak 15-30 sec after cyclic AMP addition. Phospholipid demethylation reached its maximum within 2 min and basal levels were recovered in 3 min. S-Adenosyl-L-methionine is probably the methyl donor. In vitro addition of 0.25 mM and 25MuM S-adenosyl-L-methionine to sonicated D. discoideum cells inhibited ATP-dependent 45Ca2+ uptake by 70% and 25%, respectively. Based on these lines of evidence we propose that protein and phospholipid methylation are involved in D. discoideum chemotaxis probably by regulation of intracellular Ca2+ movements.
The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes
European Journal of Biochemistry, 1987
Extracellular cAMP induces the activation of adenylate cyclase in zyxwv Dictyostelium discoideum cells. Conditions for both stimulation and inhibition of adenylate cyclase by guanine nucleotides in membranes are reported. Stimulation and inhbition were induced by GTP and non-hydrolysable guanosine triphosphates. GDP and non-hydrolysable guanosine diphosphates were antagonists. Stimulation was maximally twofold, required a cytosolic factor and was observed only at temperatures below 10 "C. An agonist of the CAMP-receptor-activated basal and GTP-stimulated adenylate cyclase 1.3-fold. Adenylate cyclase in mutant N7 could not be activated by cAMP in vivo; in vitro adenylate cyclase was activated by guanine nucleotides in the presence of the cytosolic factor of wild-type but of not mutant cells.
3':5'-Cyclic AMP-dependent 3':5'-cyclic GMP accumulation in Dictyostelium discoideum
Proceedings of the National Academy of Sciences, 1977
Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 X 108 cells per ml in 10 mM phosphate buffer, H 6.0) cAMP (5 X 10-8 M final concentration) increased cGMP levels from 1 pmol per 107 cells to 7 pmol per 107 cells in 10 sec and basal levels were recovered in 20-25 sec. cGMP accumulation did not occur when cells were in the cAMP-insensitive stage. cAMP-sensitive cells responded with increased cGMP levels when triggered by 5 X 10-8 M 5'-CH2-cAMP or
Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development
Differentiation, 1978
Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during CAMP-mediated cell aggregation in D. discoideum. During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases severalfold. In the present work we investigate the synthesis of cGMP in D. discoideum. Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum. In contrast to adenylate cyclase, which is activated by CAMP, guanylate cyclase was under our conditions activated neither by CAMP, nor by folic acid.
Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes
The Journal of biological chemistry, 1989
We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP ga...
Biochemistry, 1986
Aggregating Dictyostelium discoideum cells possess receptors for the chemoattractant cAMP on their cell surface. Membranes enriched in these receptors were isolated. Kinetic studies indicated the same receptor heterogeneity in membranes as found for intact cells [van Haastert, P. J. M., & De Wit, R. J. W. (1984) J. Biol. Chem. 259, 13321-133281. Dissociation kinetics revealed at least three receptor forms: one form, called SS, with kl = 0.9 X = 1.3 X s-l and KD = -6 nM; and one or more forms, called F, with kl > 0.1 s-'. The contribution of the SS form to the dissociation process was lower in the presence of millimolar concentrations of cAMP compared to dissociation induced by dilution only. Guanosine di-and triphosphates decreased the affinity of membranes for cAMP by increasing the dissociation rate of the CAMP-receptor complex. This was shown to result from a reduction in the number of sites of the slowly dissociating, high-affinity receptor form SS and probably also the high-affinity form S. Because the total number of cAMP binding sites was not changed by guanine nucleotides, it is inferred that the SS and S receptor forms are converted to other more rapidly dissociating receptor forms with lower affinities than SS and S. We propose that cAMP receptors in Dictyostelium membranes interact with G protein which binds guanosine di-and triphosphates. The different complexes between receptor and occupied or unoccupied G protein explain the different receptor forms and their interconversions. s-I and KD = 6.5 nM; one form, called S, with
Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase
Biochemical Journal, 2001
The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membranebound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation ; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a
Nonpolar lipid and phospholipid methylation during development of Dictyostelium discoideum
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
The life-cycle of the cellular slime mold Dictyostelium discoideum is characterized by development from a vegetative stage of solitary amoebae via cell aggregation and a 'slug' stage to a fruiting body. It has been suggested that phospholipid methylation is involved in CAMP-induced CAMP-synthesis, which is the mechanism underlying cell aggregation. Therefore, we have examined changes in lipid methylation during development. Individual amoebae incorporated the methyl group of [ methyl-3H]methionine both in phospholipid and certain nonpolar lipid components. Nonpolar methyl acceptors, characterized by thin-layer chromatography, were probably A**-stigmasten-g-01, A**-stigmasten-w-01 fatty acid ester and ubiquinone. The activities of nonpolar and phospholipid methyltransferases, as assayed in a homogenate with S-adenosyl-[methyL3H]methionine as a methyl donor, showed a strong decline from the vegetative stage to the slug stage, which was not followed by a subsequent rise during the final stage. When the activities of both enzymes were measured in intact cells during development, the decline of the amount of enzyme was masked by a 60-fold increase of the specific activity of intracellular S-adenosyl(methyL3H]methionine during cell differentiation. We conclude that the activities of phospholipid iV-methyltransferase and adenylate cyclase (or the number of cyclic AMP receptors on the cell surface) are not regulated in parallel during development.