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Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase.

ABSTRACT: 1. Cholera toxin was shown to require the presence of GTP to activate rat liver plasma-membrane adenylate cyclase. ATP did not affect the activation process. 2. Cholera toxin catalysed the incorporation of 32P from NAD labelled in the alpha-phosphate group of the ADP moiety into a rat liver plasma-membrane protein with a subunit mol.wt. of 42 500. This is taken to demonstrate ADP-ribosylation. The ADP-ribosylation of this protein also required GTP and was unaffected by ATP. 3. Nicotinamide inhibited both the activation of adenylate cyclase by cholera toxin and the ADP-ribosylation of the protein of 42 500 subunit mol wt. Neither the activation nor the ADP-ribosylation could be reversed by treatment with nicotinamide in the presence of cholera toxin.

SUBMITTER: Doberska CA

PROVIDER: S-EPMC1161710 | biostudies-other | 1980 Mar

REPOSITORIES: biostudies-other

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Cyclic adenosine 5'-diphosphate ribose analogs without a "southern" ribose inhibit ADP-ribosyl cyclase-hydrolase CD38.

Project description:Cyclic adenosine 5'-diphosphate ribose (cADPR) analogs based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesized by recently developed stereo- and regioselective N1-ribosylation. Replacing the base N9-ribose with a butyl chain generates inhibitors of cADPR hydrolysis by the human ADP-ribosyl cyclase CD38 catalytic domain (shCD38), illustrating the nonessential nature of the "southern" ribose for binding. Butyl substitution generally improves potency relative to the parent cIDPRs, and 8-amino-N9-butyl-cIDPR is comparable to the best noncovalent CD38 inhibitors to date (IC50 = 3.3 μM). Crystallographic analysis of the shCD38:8-amino-N9-butyl-cIDPR complex to a 2.05 Å resolution unexpectedly reveals an N1-hydrolyzed ligand in the active site, suggesting that it is the N6-imino form of cADPR that is hydrolyzed by CD38. While HPLC studies confirm ligand cleavage at very high protein concentrations, they indicate that hydrolysis does not occur under physiological concentrations. Taken together, these analogs confirm that the "northern" ribose is critical for CD38 activity and inhibition, provide new insight into the mechanism of cADPR hydrolysis by CD38, and may aid future inhibitor design.

| S-EPMC4207131 | biostudies-literature

Guanosine 5'-triphosphate and guanosine 5'-[beta gamma-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase.

Project description:1. GTP, but not p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, (125)I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and (125)I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.

| S-EPMC1161699 | biostudies-other

Kinetics and thermodynamics of activation of pretreatment with guanosine 5'-[beta, gamma-imido]triphosphate of smooth-muscle adenylate cyclase.

Project description:Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2 degrees C. Activation (C-->C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37 degrees C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37 degrees C. Over the range 6-24 degrees C, activation by pretreatment with p[NH]ppG followed simple Michaelis-Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased K(m) for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37 degrees C) was moderately endergonic (DeltaH(0) approximately 8kJ.mol(-1)) and accompanied by an increase in entropy (DeltaS(0) approximately 146J.mol(-1).K(-1)). The beta-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the K(m) at every temperature. This result suggested that formation of the receptor-hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the beta-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.

| S-EPMC1158475 | biostudies-other

Stabilization and solubilization of bovine corpus-luteum adenylate cyclase. The effects of guanosine triphosphate, guanosine 5'-[beta,gamma-imido]triphosphate, sodium fluoride and Tris/hydrochloric acid concentration on enzyme activity.

Project description:1. Adenylate cyclase of the washed 600g sediment of bovine corpus-luteum homogenate is stimulated by p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate), the imido analogue of GTP, and to a lesser extent by GTP itself. Activation by p[NH]ppG is not reversed by extensive washing before assay, but can, however, be reversed by NaF. 2. Both p[NH]ppG and NaF stabilize the enzyme during incubation at 37 degrees C. NaF also causes an irreversible activation, but only of part of the potentially NaF-activatable adenylate cyclase; there are possibly two components of the adenylate cyclase system, which can be distinguished by their response to NaF. 3. Solubilization of the adenylate cyclase activity in the 600g sediment, by using the non-ionic detergent Lubrol-PX, gave variable yields. A relationship between the magnitude of NaF stimulation of the 600g-sediment enzyme and the yield of soluble activity derived from the sediment was recognized. The results suggest that the pre-existing state of the enzyme complex in vivo is reflected by the response in vitro to NaF and may determine the success with which activity can be solubilized. 4. The absolute yields of soluble activity could be increased by p[NH]ppG preactivation of the 600g sediment. During the development of the maximally active state by preincubation with p[NH]ppG the enzyme passes through a stage in which Lubrol solubilization is increased, but the maximally active state is itself less amenable to solubilization. p[NH]ppG activation causes the appearance of NaF-inhibited states, which appear to be preferentially solubilized by Lubrol-PX.

| S-EPMC1184202 | biostudies-other

Evidence for regulation of human platelet adenylate cyclase by phosphorylation. Inhibition by ATP and guanosine 5'-[beta-thio]diphosphate occur by distinct mechanisms.

Project description:1. Incubation of human platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5'-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.

| S-EPMC1151050 | biostudies-other

Effects of cholera toxin and guanosine 5'-[betagamma-imido]triphosphate on beta-adrenergic-receptor affinity.

Project description:A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.

| S-EPMC1186259 | biostudies-other

Expression data from human adipocytes after adenylate cyclase activation

Project description:Progenitors in human vasculature expanded in-vitro were differentiated with adipogenic cocktail for 12 days, following which they were stimulated with forskolin for 7 days Microarrays were used to detail the program of differentiation of thermogenic human adipose cells

2018-01-22 | GSE65190 | GEO

Maintenance of endothelial guanosine triphosphate cyclohydrolase I ameliorates diabetic nephropathy.

Project description:In diabetes, endothelial nitric oxide synthase (eNOS) produces superoxide anion rather than nitric oxide, referred to as "eNOS uncoupling," which may contribute to endothelial dysfunction, albuminuria, and diabetic nephropathy. Reduced levels of endothelium-derived tetrahydrobiopterin (BH4), an essential cofactor for eNOS, promote eNOS uncoupling. Accelerated degradation of guanosine triphosphate cyclohydrolase I (GTPCH I), the rate-limiting enzyme in BH4 biosynthesis, also occurs in diabetes, suggesting that GTPCH I may have a role in diabetic microvascular disease. Here, we crossed endothelium-dominant GTPCH I transgenic mice with Ins2(+/Akita) diabetic mice and found that endothelial overexpression of GTPCH I led to higher levels of intrarenal BH4 and lower levels of urinary albumin and reactive oxygen species compared with diabetic control mice. Furthermore, GTPCH I overexpression attenuated the hyperpermeability of macromolecules observed in diabetic control mice. In addition, we treated Ins2(+/Akita) mice with metformin, which activates AMP-activated protein kinase (AMPK) and thereby slows the degradation of GTPCH I; despite blood glucose levels that were similar to untreated mice, those treated with metformin had significantly less albuminuria. Similarly, in vitro, treating human glomerular endothelial cells with AMPK activators attenuated glucose-induced reductions in phospho-AMPK, GTPCH I, and coupled eNOS. Taken together, these data suggest that maintenance of endothelial GTPCH I expression and the resulting improvement in BH4 biosynthesis ameliorate diabetic nephropathy.

| S-EPMC3699824 | biostudies-literature

Trypanosoma cruzi Phosphomannomutase and Guanosine Diphosphate-Mannose Pyrophosphorylase Ligandability Assessment.

Project description:Chagas' disease, which is caused by the Trypanosoma cruzi parasite, has become a global health problem that is currently treated with poorly tolerated drugs that require prolonged dosing. Therefore, there is a clinical need for new therapeutic agents that can mitigate these issues. The phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GDP-MP) enzymes form part of the de novo biosynthetic pathway to the nucleotide sugar GDP-mannose. This nucleotide sugar is used either directly, or indirectly via the formation of dolichol-phosphomannose, for the assembly of all mannose-containing glycoconjugates. In T. cruzi, mannose-containing glycoconjugates include the cell-surface glycoinositol-phospholipids and the glycosylphosphatidylinositol-anchored mucin-like glycoproteins that dominate the cell surface architectures of all life cycle stages. This makes PMM and GDP-MP potentially attractive targets for a drug discovery program against Chagas' disease. To assess the ligandability of these enzymes in T. cruzi, we have screened 18,117 structurally diverse compounds exploring drug-like chemical space and 16,845 small polar fragment compounds using an assay interrogating the activities of both PMM and GDP-MP enzymes simultaneously. This resulted in 48 small fragment hits, and on retesting 20 were found to be active against the enzymes. Deconvolution revealed that these were all inhibitors of T. cruzi GDP-MP, with compounds 2 and 3 acting as uncompetitive and competitive inhibitors, respectively. Based on these findings, the T. cruzi PMM and GDP-MP enzymes were deemed not ligandable and poorly ligandable, respectively, using small molecules from conventional drug discovery chemical space. This presents a significant hurdle to exploiting these enzymes as therapeutic targets for Chagas' disease.

| S-EPMC6761512 | biostudies-literature

Efficient enzymatic synthesis of guanosine 5'-diphosphate-sugars and derivatives.

Project description:An N-acetylhexosamine 1-kinase from Bifidobacterium infantis (NahK_15697), a guanosine 5'-diphosphate (GDP)-mannose pyrophosphorylase from Pyrococcus furiosus (PFManC), and an Escherichia coli inorganic pyrophosphatase (EcPpA) were used efficiently for a one-pot three-enzyme synthesis of GDP-mannose, GDP-glucose, their derivatives, and GDP-talose. This study represents the first facile and efficient enzymatic synthesis of GDP-sugars and derivatives starting from monosaccharides and derivatives.

| S-EPMC3915774 | biostudies-literature

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