Guanine Nucleotide Sites Research Articles

Is serotonin transporter genotype associated with epigenetic susceptibility or vulnerability? Examination of the impact of socioeconomic status risk on African American youth

We hypothesized that presence of the short allele in the promoter region of the serotonin transporter would moderate the effect of early cumulative socioeconomic status (SES) risk on epigenetic change among African American youth. Contrasting hypotheses regarding the shape of the interaction effect were generated using vulnerability and susceptibility frameworks and applied to data from a sample of 388 African American youth. Early cumulative SES risk assessed at 11-13 years based on parent report interacted with presence of the short allele to predict differential methylation assessed at age 19. Across multiple tests, a differential susceptibility perspective rather than a diathesis-stress framework best fit the data for genes associated with depression, consistently demonstrating greater epigenetic response to early cumulative SES risk among short allele carriers. A pattern consistent with greater impact among short allele carriers also was observed using all cytosine nucleotide-phosphate-guanine nucleotide sites across the genome that were differentially affected by early cumulative SES risk. We conclude that the short allele is associated with increased responsiveness to early cumulative SES risk among African American youth, leading to epigenetic divergence for depression-related genes in response to exposure to heightened SES risk among short allele carriers in a "for better" or "for worse" pattern.

A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure.

An updated topological model is constructed for the catalytic nucleotide-binding site of the F1-ATPase. The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and the ras p21 protein. Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the beta subunit of F1. Several potentially important residues on beta are noted that have not yet been studied by mutagenesis or affinity labeling.

Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc − variants are prevented by islet-activating protein

cyc −-Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc − cells with islet-activating protein (IAP), which causes ADP-ribosylation of an M r 40 000 polypeptide in cyc − membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTPγS. Furthermore, somatostatin-induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.

Occurrence of a hormone-sensitive inhibitory coupling component of the adenylate cyclase in S49 lymphoma cyc- variants.

The influence of somatostatin was studied on cyclic AMP levels and adenylate cyclase activity in cyc- variants of S49 lymphoma cells. These cells are deficient in the guanine nucleotide site that mediates hormone-induced adenylate cyclase stimulation, but their cyclase can be stimulated by forskolin. Somatostatin maximally decreased the 30 microM forskolin-stimulated cyclic AMP levels by 35%. Half-maximal suppression occurred at about 0.1 nM somatostatin. Somatostatin (up to 1 microM) had no effect on the 100 microM forskolin-stimulated adenylate cyclase activity in cyc- membrane preparations when guanine nucleotides were not present. In the presence of GTP, however, which by itself caused a small decrease in activity, somatostatin maximally inhibited the enzyme by 20-25%. GTP was half-maximally effective at 0.1 microM, and half-maximal inhibition by somatostatin was observed at 0.1- 1 nM. In the presence of the stable GTP analog guanosine 5'-O-(3-thiotriphosphate) (1 microM), which decreased the stimulated activity by about 40% after a short lag period, somatostatin (1 microM) did not cause a further decrease in final activity but reduced the lag period by about 50%. The data indicate that membranes of cyc- variants contain a regulatory site that mediates both guanine nucleotide and hormone-induced inhibition of the adenylate cyclase and suggest that the mechanisms of activation and inactivation of this inhibitory site are similar to those of the stimulatory component missing in cyc-membranes.

A nucleotide regulatory site for somatostatin inhibition of adenylate cyclase in S49 lymphoma cells.

The cyc- variants of S49 lymphoma cells have served as powerful tools for studying the components and mechanisms of hormone-induced adenylate cyclase stimulation, as these cells are deficient in the guanine nucleotide regulatory site (Ns) mediating hormone, guanine nucleotide, cholera toxin and fluoride-induced stimulations of the enzyme. Because of this deficiency, membranes of these cells have been used for reconstitution of the system by inserting the coupling component derived from other cell types. The hormone-sensitive adenylate cyclase is not only stimulated by hormones but can also be inhibited by a wide variety of hormones and neurotransmitters, and there is some evidence that hormonal inhibition may be mediated by a distinct guanine nucleotide regulatory site. Studies in cyc- cells lacking a functional Ns may therefore answer this unresolved, important question. We have recently observed that stable GTP analogues can inhibit cyc- adenylate cyclase stimulated by purified, preactivated Ns or forskolin, which can activate adenylate cyclase even in the absence of a functional Ns (ref. 10). The data indicated that these Ns-deficient cells contain an inhibitory guanine nucleotide site, Ni. To strengthen this concept, we investigated whether the cyc- adenylate cyclase can be inhibited by a hormone. We report here that somatostatin decreases cyclic AMP levels in cyc- cells, inhibits the forskolin-stimulated adenylate cyclase and causes a concomitant increase in a high affinity GTPase activity in cyc- membranes. The data strongly suggest that both the hormone- and guanine nucleotide-induced adenylate cyclase inhibitions in cyc- cells are mediated by Ni and that the mechanisms of activation and inactivation of Ni are similar to those established for Ns.

Allosteric changes of the guanine nucleotide site of elongation factor EF-Tu: A comparative study of two kirromycin-resistant EF-Tu species

Allosteric changes of the guanine nucleotide site of elongation factor EF-Tu: A comparative study of two kirromycin-resistant EF-Tu species

Inactivation of the guanine nucleotide regulatory site mediating inhibition of the adenylate cyclase in hamster adipocytes.

The influence of N-ethylmaleimide and trypsin was studied on stimulatory and inhibitory regulations of the hamster adipocyte adenylate cyclase. Treatment of intact adipocytes or adipocyte ghosts with N-ethylmaleimide decreased basal and forskolin-stimulated adenylate cyclase activities. In the pretreated membrane preparations, inhibition of the enzyme by GTP and by stable GTP analogues was abolished. Concomitantly, activation of the adenylate cyclase by NaCl and its inhibition by the antilipolytic agents, prostaglandin E1 and nicotinic acid, were obliterated. In contrast, adenylate cyclase stimulation by ACTH and stable GTP analogues was not impaired but rather increased. Similarly, the NaCl-induced attenuation of the ACTH-stimulated enzyme activity was increased by the N-ethylmaleimide treatment. Limited proteolysis of hamster adipocyte ghosts with trypsin also obliterated GTP and prostaglandin E1-induced inhibitions and NaCl-induced activation of the adenylate cyclase. In contrast, adenylate cyclase activity stimulated by isoproterenol was increased after trypsin treatment. The data suggest that the activity of the adenylate cyclase is regulated via two distinct guanine nucleotide sites and that treatment with N-ethylmaleimide and limited proteolysis with trypsin functionally eliminates the regulatory site mediating adenylate cyclase inhibition, leading to a state where the enzyme activity is regulated only via the stimulatory site. The differential effects of these treatments on NaCl-induced activation and attenuation of the adenylate cyclase suggest that sodium acts on both regulatory sites in an inhibitory manner, and that by the functional elimination of the inhibitory site, only the sodium-induced attenuation of the adenylate cyclase via the stimulatory site is observed.

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E 1, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn 2+ and Mg 2+, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn 2+ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E 1 (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn 2+, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E 1 − , nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn 2+, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn 2+ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg 2+, although about 100-fold higher concentrations of Mg 2+ than of Mn 2+ were required. The data indicate that Mn 2+at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn 2+ on the functions of the guanine nucleotide site(s) in the system.

Interaction between beta-adrenergic receptors and guanine nucleotide sites in turkey erythrocyte membranes.

beta 1-Adrenergic receptors from turkey erythrocyte membranes have been identified by specific binding of the radiolabeled antagonist (-)-[3H]dihydroalprenolol. These receptors are inactivated by the alkylating agent N-ethylmaleimide when occupied by beta-adrenergic agonists but not when occupied by antagonists or when unoccupied. A time-dependent decrease of the number of receptor sites is observed. Inactivation affects 45-60% of the sites, regardless of the agonist or N-ethylmaleimide concentration. The guanine nucleotides GTP and 5'-guanylyl imidodiphosphate effectively protect the receptors against agonist-mediated inactivation by N-ethylmaleimide. Protection by ATP necessitates a 100-fold higher concentration; 10 mM NaF is ineffective. The guanine nucleotide effect is reversible and occurs via interaction with N-ethylmaleimide-insensitive sites. These observations establish that guanine nucleotide sites interact with and caused structural modification of the agonist-occupied beta-adrenergic receptors in turkey erythrocyte membranes.

Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1, and guanine nucleotides. Evidence for separate guanine nucleotide sites mediating stimulation and inhibition.

A method for preparing human platelet membranes with high adenylate cyclase activity is described. Using these membranes, epinephrine and GTP individually are noted to inhibit adenylate cyclase slightly. When present together, epinephrine and GTP act synergistically to cause a 50% inhibition of basal activity. The epinephrine effect is an alpha-adrenergic process as it is reversed by phentolamine but not propranolol. The quasi-irreversible activation of adenylate cyclase by Gpp(NH)p is time, concentration, and Mg2+-dependent but is not altered by the presence of epinephrine. Adenylate cyclase activated by Gpp(NH)p, and extensively washed to remove unbound Gpp(NH)p, is inhibited by the subsequent addition of Gpp(NH)p, GTP, and epinephrine. This effect of epinephrine is also an alpha-adrenergic phenomenon. In contrast to epinephrine which inhibits the cyclase, PGE1 addition results in enzyme stimulation. PGE1 stimulation does not require GTP addition. PGE1 accelerates the rate of Gpp(NH)p-induced activation. Low GTP concentrations (less than 1 x 10(-6) M) enhance PGE1 stimulation while higher GTP concentrations cause inhibition. These observations suggest that human platelet adenylate cyclase possesses at least two guanine nucleotide sites, one which interacts with the alpha-receptor to result in enzyme inhibition and a second guanine nucleotide site which interacts with the PGE1 receptor and causes enzyme stimulation.

Evidence for distinct guanine nucleotide sites in the regulation of the glucagon receptor and of adenylate cyclase activity.

Evidence for distinct guanine nucleotide sites in the regulation of the glucagon receptor and of adenylate cyclase activity.

Binding sites for calcium on tubulin.

Calcium ions can inhibit the in vitro assembly of microtubules and, therefore, may play a role in the regulation of microtubule formation in vivo. In order to test the validity of this hypothesis; the interaction between calcium and pruified brain microtubular protein has been investigated by standard binding assays. We have detected and characterized two classes of binding sites for calcium on tubulin, the major component of cytoplasmic microtubules. There is a single high-affinity site per tubulin molecule, characterized by a dissociation constant of 3.2 X 10(-6) M. That site is inhibited by magnesium (k1 = 5 X 10(-5) M) and potassium chloride. There are approximately 16 low-affinity sites which have a dissociation constant of 2.8 X 10(-4) M, and which are also inhibited by potassium chloride. Binding at the low-affinity sites is slightly enhanced by low magnesium concentrations. Both classes of sites are distinguishable from the colchicine binding site, and are apparently also distinct from the vinblastine and guanine nucleotide sites. The characteristics of the calcium binding activity of tubulin are similar to those found for the calcium-binding proteins of sarcoplasmic reticulum. The results are consistent with a physiological role for calcium in the regulation of microtubule assembly.

Neurotubule assembly at substoichiometric nucleotide levels using a GTP regenerating system

Tubulin derived from cold depolymerized bovine microtubules has been gel filtered to obtain a tubulin preparation with only 3% of the tubulin dimers containing exchangeable [ 3H]-guanine nucleotide. In the presence of acetyl-P and bacterial acetate kinase, this preparation polymerizes to form microtubules which are morphologically indistinguishable from microtubules formed in the presence of excess GTP. The extent of microtubule formation at substoichiometric nucleotide levels using the GTP regenerating system exceeds the extent of assembly obtained with excess GTP. It is concluded that the exchangeable guanine nucleotide site can be virtually unoccupied in intact neurotubules and this finding indicates that GDP can “catalyze” tubule assembly in the presence of a GTP regenerating system.