Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.
Ontology highlight
ABSTRACT:
SUBMITTER: Sulakhe PV
PROVIDER: S-EPMC1164758 | biostudies-other | 1977 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.
| S-EPMC1147565 | biostudies-other
Project description:In the present study, we demonstrate that AC5 (type V adenylate cyclase) interacts with Ric8a through directly interacting at its N-terminus. Ric8a was shown to be a GEF (guanine nucleotide exchange factor) for several alpha subunits of heterotrimeric GTP binding proteins (Galpha proteins) in vitro. Selective Galpha targets of Ric8a have not yet been revealed in vivo. An interaction between AC5 and Ric8a was verified by pull-down assays, co-immunoprecipitation analyses, and co-localization in the brain. Expression of Ric8a selectively suppressed AC5 activity. Treating cells with pertussis toxin or expressing a dominant negative Galphai mutant abolished the suppressive effect of Ric8a, suggesting that interaction between the N-terminus of AC5 and a GEF (Ric8a) provides a novel pathway to fine-tune AC5 activity via a Galphai-mediated pathway.
| S-EPMC2049038 | biostudies-literature
Project description:1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a ;burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a ;lag' phase with p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.
| S-EPMC1163311 | biostudies-other
Project description:1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30 degrees C in response to p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0 degrees C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.
| S-EPMC1161759 | biostudies-other
Project description:BackgroundAutism is a neurodevelopmental disorder affecting sociocommunicative behavior, but also sensorimotor skill learning, oculomotor control, and executive functioning. Some of these impairments may be related to abnormalities of the caudate nuclei, which have been reported for autism.MethodsOur sample was comprised of 8 high-functioning males with autism and 8 handedness, sex, and age-matched controls. Subjects underwent functional MRI scanning during performance on simple visuomotor coordination tasks. Functional connectivity MRI (fcMRI) effects were identified as interregional blood oxygenation level dependent (BOLD) signal cross-correlation, using the caudate nuclei as seed volumes.ResultsIn the control group, fcMRI effects were found in circuits with known participation of the caudate nuclei (associative, orbitofrontal, oculomotor, motor circuits). Although in the autism group fcMRI effects within these circuits were less pronounced or absent, autistic subjects showed diffusely increased connectivity mostly in pericentral regions, but also in brain areas outside expected anatomical circuits (such as visual cortex).ConclusionThese atypical connectivity patterns may be linked to developmental brain growth disturbances recently reported in autism and suggest inefficiently organized functional connectivity between caudate nuclei and cerebral cortex, potentially accounting for stereotypic behaviors and executive impairments.
| S-EPMC1635430 | biostudies-literature
Project description:Monoclonal antibodies (Mabs) to the stimulatory (Ns) and inhibitory (Ni) guanine nucleotide regulatory proteins associated with adenylate cyclase have been developed. Two Mabs (2A3 and 5G12), which are of the IgG2b subclass, recognize the beta-subunits (beta) of Ns, Ni and transducin. Iodinated beta can be immunoprecipitated by either Mab coupled to Affi-Gel 10 and this can be decreased by prior incubation of the Mabs with excess unlabelled beta. The Mabs stabilize the activated state of Ns while decreasing the rate of deactivation of activated Ns in the presence of beta.
| S-EPMC1146815 | biostudies-other
Project description:Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.
| S-EPMC1135034 | biostudies-other
Project description:Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.
| S-EPMC1161602 | biostudies-other
Project description:RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I-III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines.
| S-EPMC8486122 | biostudies-literature
Project description:1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.
| S-EPMC1165744 | biostudies-other