Requirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin.

The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible to assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000--20,000.     

Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase.

Studies on the reaction kinetics and chromatographic properties of detergent-dispersed adenylate cyclase are described. Detergent-dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver. Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP--4 (or HATP--3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg++ and Mn++ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP--3 or ATP--4. The detergent-dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE-Sephadex but was not required when DEAE-agarose was used. Dispersed brain cyclase was also chromatographed on agarose-NH(CH2)3NH(CH2)3-NH2 which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent-dispersed adenylate cyclase may reflect the influence of both hydrophobic and ionic factors.     

Vasopressin-sensitive kidney adenylate cyclase: modulation of the hormonal response.

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg2+, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg2+, and GMPPNP, respectively. Mg2+ ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg2+ interacts with an enzyme regulatory site. Finally, Mg2+ can modulate the hormonal response, with Mg2+ ions affecting the coupling function--that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg2+ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg2+ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30 degrees C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15 degrees C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10(-8)M to 10(-5)M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg2+ concentrations except for the increase in velocity when raising Mg2+ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland.

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodiphosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

Mechanism of action of choleragen.

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.     

Use of 13C NMR spectrometric data to produce a predictive model of estrogen receptor binding activity

We have developed a spectroscopic data-activity relationship (SDAR) model based on 13C NMR spectral data for 30 estrogenic chemicals whose relative binding affinities (RBA) are available for the alpha (ERalpha) and beta (ERbeta) estrogen receptors. The SDAR models segregated the 30 compounds into strong and medium binding affinities. The SDAR model gave a leave-one-out (LOO) cross-validation of 90%. Two compounds that were classified incorrectly in the SDAR model were in the transition zone between classifications. Real and predicted 13C NMR chemical shifts were used with test compounds to evaluate the predictive behavior of the SDAR model. The 13C NMR SDAR model using predicted 13C NMR data for the test compounds provides a rapid, reliable, and simple way to screen whether a compound binds to the estrogen receptors.     

Effect of essential fatty acid deficiency on activity of liver plasma membrane enzymes in the rat.

Liver plasma membranes (LPM) were isolated from rats fed an essential fatty acid-supplemented diet (+EFA) or from rats fed an essential fatty acid-deficient diet (-EFA). The proportions of linoleate and arachidonate in membrane total fatty acids in the -EFA preparations were one-half or less than the values for the +EFA preparations. Basal, F-, or glucagon-stimulated adenylate cyclase activities were significantly lower in EFA-deficient livers than in nondeficient ones. Addition of GTP significantly enhanced glucagon-stimulated adrenylate cyclase in both groups, but extent of stimulation above basal was greater in EFA-deficient livers. Portal vein injection of glucagon in vivo resulted in significantly higher cAMP formation in +EFA livers than in -EFA livers. When glucagon was used in vitro at 1-1,000 nM, stimulation of adenylate cyclase remained lower in EFA-deficient membranes, but extent of stimulation above basal activity was larger in -EFA membranes than in +EFA. Total Na+, K+ (Mg2+)-ATPase from EFA-depleted LPM exhibited significantly higher values of apparent Km and Vmax-5'-Nucleotidase activity, in contrast, was considerably decreased in EFA-deficient rats. These findings show that, in animals, changes in unsaturated fatty acid composition can affect the properties of membrane-bound enzymes. These alterations could be due to changes in membrane physical properties and/or prostaglandin formation.     

The Role of Triazole and Glucose Moieties in Alkali Metal Cation Complexation by Lower-Rim Tertiary-Amide Calix[4]arene Derivatives

The binding of alkali metal cations with two tertiary-amide lower-rim calix[4]arenes was studied in methanol, N,N-dimethylformamide, and acetonitrile in order to explore the role of triazole and glucose functionalities in the coordination reactions. The standard thermodynamic complexation parameters were determined microcalorimetrically and spectrophotometrically. On the basis of receptor dissolution enthalpies and the literature data, the enthalpies for transfer of reactants and products between the solvents were calculated. The solvent inclusion within a calixarene hydrophobic basket was explored by means of ¹H NMR spectroscopy. Classical molecular dynamics of the calixarene ligands and their complexes were carried out as well. The affinity of receptors for cations in methanol and N,N-dimethylformamide was quite similar, irrespective of whether they contained glucose subunits or not. This indicated that sugar moieties did not participate or influence the cation binding. All studied reactions were enthalpically controlled. The peak affinity of receptors for sodium cation was noticed in all complexation media. The complex stabilities were the highest in acetonitrile, followed by methanol and N,N-dimethylformamide. The solubilities of receptors were greatly affected by the presence of sugar subunits. The medium effect on the affinities of calixarene derivatives towards cations was thoroughly discussed regarding the structural properties and solvation abilities of the investigated solvents.     

The Escherichia coli adenylate cyclase complex: activation by phosphoenolpyruvate.

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

PACAP and its receptors exert pleiotropic effects in the nervous system by activating multiple signaling pathways

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from the ovine brain in 1989 as a novel hypothalamic hormone that potently activates adenylate cyclase to produce cyclic AMP in pituitary cells. This neuropeptide belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) superfamily, and exists in two amidated forms as PACAP38 (38-amino acid residues) and PACAP27 derived from the same precursor. The primary structure of PACAP has been remarkably conserved throughout evolution among tunicata, ichthyopsida, amphibia and mammalia, and a PACAP-like neuropeptide has also been determined in Drosophila. Both PACAP and its receptors are mainly distributed in the nervous and endocrine systems showing pleiotropic functions with high potency. There are three types of receptors with high PACAP-binding affinity and with different tissue-distribution patterns. All of them belong to G-protein-coupled receptor superfamily with seven transmembrane domains. PAC(1) is the PACAP-specific receptor and exists in at least eight splice variants which couple to different intracellular signal transduction pathways. VPAC(1) and VPAC(2) are the common receptors for both PACAP and VIP, which are coupled to adenylate cyclase. This review article presents and discusses an update on PACAP research and its pleiotropic physiological functions based on multiple receptor-mediated signaling mechanisms in both the central and peripheral nervous system, including the regulation of hypothalamic neurosecretion, homeostatic control of circadian clock and behavioral actions, involvement in learning and memory processes, neuroprotective effects such as anti-apoptosis and response to injury and inflammation, and neural ontogenetic functions on proliferation/differentiation processes from early stages.     

Beta-adrenergic receptors: regulatory role of agonists.

Direct radioligand binding studies have been used to probe the molecular mechanisms whereby agonist catecholamines regulate the function of beta-adrenergic receptors in a model system, the frog erythrocyte. The unique characteristics of agonist as opposed to antagonist action are first, the ability to stimulate the adenylate cyclase through the receptor and second, the ability to desensitize the system by alterations induced in beta-adrenergic receptors. These properties of agonist are not shared by antagonist despite the high affinity and specificity of antagonist binding to the beta-adrenergic receptors. Agonist and antagonist receptor complexes may be distinguished in a variety of ways including differences in their sensitivity to regulatory guanine nucleotides and also by gel chromatography on AcA 34 Ultragel. The agonist receptor complex appears to elute from the columns with an apparently increased size. A "dynamic receptor affinity model" of beta-adrenergic receptor action is proposed which features several distinct conformational states of the receptor. Agonists have much higher affinity for the physiologically active or coupled state of the receptor, whereas antagonists have equal affinity for both. In addition, a third "desensitized" state of the receptor is also postulated to exist.     

Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes

Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase beta during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase beta in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1beta was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes.     

862 — The sequence and energetics of cell membrane transductive coupling to intracellular enzyme systems

We have shown that weak oscillating electromagnetic fields in the pericellular environment modulate key steps in coupling of signals from humoral stimuli at cell surface receptors to intracellular systems. This paper summarizes evidence that enzymatic activity within the cell provides sensitive molecular markers about both the sequence and the energetics of transmembrane coupling mechanisms. As research tools, these imposed fields appear to offer unique opportunities for understanding highly non-linear, non-equilibrium aspects of these coupling mechanisms, including the basis for amplification of weak pericellular stimuli to achieve an energetic threshold in signaling to intracellular enzyme systems.A three-stage model of membrane transductive coupling is proposed: a first stage in which weak pericellular electrochemical oscillations and binding of humoral stimulating molecules at receptor sites initiates a highly cooperative modification of calcium binding, a second stage involving transmission of signals initiated at receptor sites to the cell interior; and a third stage dealing with intracellular response to the transmembrane signal.Low frequency pulsed magnetic fields modulate stimulation of adenylate cyclase by parathyroid hormone (PTH) in bone cells. From collateral studies of field effects on PTH-binding to its receptor and from studies of fluoride activation of adenylate cyclase, there is evidence that an important field action is on membrane coupling proteins between receptors for PTH and adenylate cyclase.In addition to an action on adenylate cyclase, weak pericellular fields modulate activity of messenger enzymes, the protein kinases, and of an enzyme essential for cell growth, omithine decarboxylase. In human lymphocytes, cAMP-independent protein kinases are transiently inhibited by exposure to weak (athermal) microwave fields sinusoidally modulated at 16 Hz. In liver and ovary cells exposed to the same fields, and in bone cells exposed to low frequency pulsed magnetic fields, omithine decarboxylase activity is increased.Experimental data and models interrelating the pericellular electrochemical environment, cancer-promoting phorbol esters and activities of protein kinases and ornithine decarboxylase are discussed.     

Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP phosphodiesterase: a specific role for a light-sensitive GTPase as a component in the activation sequence.

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3'5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.     

Synthesis of N-octylmonoaza-15-crown-5 having a dodecylphosphoric acid functional group and the binding ability toward alkali metal cations

A new N-octylmonoaza-15-crown-5 having an alkylphosphoric acid functional group ( 3c ) was synthesized. It was revealed that 3c selectively transported sodium ion under neutral source phase/acidic receiving phase condition, and selectively transported lithium ion under basic source phase/acidic receiving phase condition. From an ir and ¹³C nmr spectral study of the lithium hydroxide and sodium thiocyanate complexes of 3c , it is suggested that 3c does not incorporate cations into the three dimensional cavity using the crown ring and the phosphoric acid site, but that the crown and the phosphoric acid sites act on the cations independently; only the crown ether site of 3c significantly coordinates to the cations under neutral source phase condition, and the phosphate anion is mainly employed under basic conditions.     

Adsorptive bubble separation of zinc and cadmium cations in presence of ferric and aluminum hydroxides

The adsorptive bubble separation of zinc and cadmium cations from solution in the presence of ferric and aluminum hydroxides was carried out by means of Tween 80 (nonionic surfactant), and sodium laurate and stearate (anionic surfactants). The mechanism of metal removal is different depending on the nature of the surfactant used. The removal of zinc cations by adsorbing colloid flotation is higher than that of cadmium cations. It increases with increases in the amount of hydroxide precipitate and the concentration of Tween 80. The removal of zinc cations by ion flotation is lower than that of cadmium cations. It does not change with increases in the hydroxide amount. It increases, however, with increased sodium laurate or stearate concentration. Both separation methods turned out to be helpful for studying both the solution's structure and the interactions at the solution-solid interface.     

Calculated hydride and fluoride affinities of a series of carbenium and silylium cations in the gas phase and in C6H5Cl solution

We report the hydride and fluoride affinities for a group of silylium and carbenium cations. With comparable substituents on the central atom, the silylium cations have the higher fluoride affinity, whereas the carbenium ions have the higher hydride affinity. In the first approximation, the hydride and the fluoride affinities vary in parallel with changes in substitution, but the deviations from linear correspondence of hydride and fluoride affinities are more pronounced for carbenium ions. The hydride and fluoride affinities of silylium cations are very similar, whereas for carbenium ions, the hydride affinities are 35–60 kcal mol^?1 higher than fluoride affinities. These results are placed in the context of studies of hydrodefluorination of aliphatic C? F bonds enabled by silylium carborane catalysts [C. Douvris, O. V. Ozerov, Science 2008 , 321, 1188]. The abstraction of fluoride from perfluoroalkanes by a trialkylsilylium cation is neither thermodynamically favorable nor kinetically accessible and, if at all possible, will require a much more fluorophilic silylium cation.     

Selective recognition of alkyl pyranosides in protic and aprotic solvents

The design and synthesis of receptors capable of selective, noncovalent recognition of carbohydrates continues to be a signature challenge in bioorganic chemistry. We report a new generation of tripodal receptors incorporating three pyridine (compound 2) or quinoline (compound 3) rings around a central cyclohexane core for use in molecular recognition of monosaccharides in apolar and polar protic solvents. These tripodal receptors were investigated using (1)H NMR, UV, and fluorescence titrations in order to determine their binding abilities toward a set of octyl glycosides. Receptor 2 displayed the highest binding affinity reported to date for noncovalent 1:1 binding of an alpha-glucopyranoside in chloroform (Ka = 212,000 +/- 27,000 M(-1)) and an approximately 8-fold selectivity for the alpha anomer over the beta anomer of the glucopyranoside. Most importantly, 2 retained its micromolar range of affinities toward monosaccharides in a polar and highly competitive solvent (methanol). The quinoline variant 3 also displayed micromolar binding affinities for selected monosaccharides in methanol (as measured by fluorescence) that were generally smaller than those of 2. Compound 3 was found to follow a selectivity pattern similar to that of 2, displaying higher affinities for glucopyranosides than for other monosaccharides. The binding stoichiometry was estimated to be 1:1 for the complexes formed by both 2 and 3 with glucopyranosides, as determined by Job plots. Nuclear Overhauser effect spectroscopy allowed for the derivation of a binding model consistent with the observed selectivities.     

Vinylphosphonic esters as end-capping agents in group transfer polymerization

Diethyl vinylphosphonate does not undergo group transfer polymerization (GTP), but does react with the silyl ketene acetal end group of PMMA prepared by GTP to give α-(2-diethoxyphosphinylethyl) PMMA. Copolymerization of MMA and small amounts of diethyl vinylphosphonate led to copolymer. The telechelic PMMA diphosphonic acid, α-(2-dihydroxyphosphinylethyl) ω-dihydroxyphosphinylPMMA, was synthesized by initiation of GTP of MMA with diethyl 3-methoxy-3-trimethylsiloxy-2-propene-1-phosphonate, followed by termination with diethyl vinylphosphonate, silylation of the phosphonic ester with bromotrimethylsilane, and hydrolysis. Reaction of living poly (methyl methacrylateco-n-butyl methacrylate), prepared by GTP, with bis (trimethylsilyl) vinylphosphonate followed by hydrolysis gave α-(2-dihydroxyphosphinylethyl) poly (methyl methacrylateco-n-butyl methacrylate).