10 research outputs found
Regulatory Properties of Magnesium-dependent Guanylate Cyclase in Dictyostelium discoideum Membranes
Evolving minimum standards in responsible international sperm donor offspring quota
An international working group was established with the aim of making recommendations on the number of offspring for a sperm donor that should be allowable in cases of international use of his sperm. Considerations from genetic, psychosocial, operational and ethical points of view were debated. For these considerations, it was assumed that current developments in genetic testing and Internet possibilities mean that, now, all donors are potentially identifiable by their offspring, so no distinction was made between anonymous and non-anonymous donation. Genetic considerations did not lead to restrictive limits (indicating that up to 200 offspring or more per donor may be acceptable except in isolated social-minority situations). Psychosocial considerations on the other hand led to proposals of rather restrictive limits (10 families per donor or less). Operational and ethical considerations did not lead to more or less concrete limits per donor, but seemed to lie in-between those resulting from the aforementioned ways of viewing the issue. In the end, no unifying agreed figure could be reached; however the consensus was that the number should never exceed 100 families. The conclusions of the group are summarized in three recommendation
The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes
Extracellular cAMP induces the activation of adenylate cyclase in 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.
Preincubation of membranes under phosphorylation conditions has been shown to alter the interaction between cAMP receptor and G protein. These phosphorylation conditions converted stimulation to inhibition of adenylate cyclase by guanine nucleotides. Inhibition was maximally 30% and was not affected by the cytosolic factor involved in stimulation.
In membranes obtained from cells that were treated with pertussis toxin, adenylate cyclase stimulation by guanine nucleotides was as in control cells, whereas inhibition by guanine nucleotides was lost. When cells were desensitized by exposure to cAMP agonists for 15 min, and adenylate cyclase was measured in isolated membranes, stimulation by guanine nucleotides was lost while inhibition was retained. These results suggest that Dictyostelium discoideum adenylate cyclase may be regulated by Gs-like and Gi-like activities, and that the action of Gs but not Gi is lost during desensitization in vivo and by phosphorylation conditions in vitro.
Forms of the Chemotactic Adenosine 3’,5’-Cyclic Phosphate Receptor in Isolated Dictyostelium discoideum Membranes and Interconversions Induced by Guanine Nucleotides
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. Dissociation kinetics revealed at least three receptor forms: one form, called SS, with k-1 = 0.9 × 10-3 s-1 and KD = 6.5 nM; one form, called S, with k-1 = 1.3 × 10-2 s-1 and KD = ~6 nM; and one or more forms, called F, with k-1 > 0.1 s-1. 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.
Regulatory Properties of Magnesium-dependent Guanylate Cyclase in Dictyostelium discoideum Membranes
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 µM guanosine 5’-O-(3-thio)triphosphate (GTPγS) or 100 µM guanosine 5’-(β,γ-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’-(β,γ-imido)triphosphate. 2) Both basal and GTPγS-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTPγS to stimulate the enzyme after cell lysis. 3) Inclusion of 25 µM GTPγS during cell lysis reduced the KM for GTP from 340 to 85 µM 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, GTPγS was also a substrate for the enzyme with a KM = 120 µM and a Vmax = 115 pmol/min∙mg protein. 5) GTPγS-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 × g; however, only sedimentable enzyme was stimulated by GTPγS. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.
G-protein-mediated Interconversions of Cell-surface cAMP Receptors and Their Involvement in Excitation and Desensitization of Guanylate Cyclase in Dictyostelium discoideum
G-protein-mediated Interconversions of Cell-surface cAMP Receptors and Their Involvement in Excitation and Desensitization of Guanylate Cyclase in Dictyostelium discoideum
In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale.
Statistical analysis of the association of [3H]cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme.
BF→BS→BSS
cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t½ = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein.
Desensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state.
These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition.