The hormone-sensitive adenylyl cyclase system of the ciliate Dileptus anser

The hormone-sensitive adenylyl cyclase system (AC system) was found and characterized for unicellular eukaryotes--the ciliatae Dileptus anser. It has been first shown that hormones of higher eukaryotes--biogenic amines (adrenalin, isoproterenol and serotonin) and peptide glucagon--stimulate in dose-dependent manner the activity of adenylyl cyclase (AC) of D. anser. The enzymatic activity was stimulated also by guanine nucleotides--GTP and their non-hydrolysable analogue Gpp[NH]p. Stimulating effects of hormones and guanine nucleotides strongly depend on the level of AC basal activity, which is relatively easy to reach (1430 to 3900 pmol cAMP/min per 1 mg of protein). The sensitivity of D. anser AC system to hormones and guanine nucleotides shows the presence of receptor or receptor-related molecules, capable of interacting with the hormone and activating AC through heterotrimeric G-proteins, in ciliatae. On the base of obtained data, a conclusion is made about the similarity of the structural-functional organization of AC systems of D. anser and higher eukaryotes.     

Modulation of adenylate cyclase by guanine nucleotides and Kirsten sarcoma virus mediated transformation

Certain tumour cells contain activated ras genes that code for 21 000 dalton proteins (p21). These proteins associate with the inner face of the plasma membrane and bind guanine nucleotides specifically. In order to determine whether p21s have functions similar to other GTP binding proteins, we investigated the regulation, by guanine nucleotides, of adenylate cyclase (AC) activity in membrane preparations isolated from fibroblasts (C127) transformed by a temperature sensitive mutant of Kirsten sarcoma virus (Ts 371). The degree of AC stimulation by GMP P(NH)P increased when these cells were shifted from the permissive temperature (33 degrees C) to the non-permissive temperature (39 degrees C). This effect was more pronounced at low Mg++ and low GMP P(NH)P concentrations. AC stimulation remained unchanged in rat fibroblasts infected with a temperature sensitive mutant of Rous Sarcoma virus. AC activity was depressed in C127 cells infected with wild type KiMSV. Our data illustrate the feasibility of correlating alterations in the AC system with ras gene expression and using such experimental approaches to elucidate the physiological functions of the p21 proteins.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

A transplantable rat Leydig cell tumor--3. Isoproterenol and prostaglandin E1 (PGE1) responsive adenylyl cyclase (AC). Regulatory effects of nucleotides and magnesium

The activity of the membrane bound adenylyl cyclase (AC), the effects of nucleotides, Mg2+-cations and its responsiveness to isoproterenol and prostaglandin E1 (PGE1) were examined in a transplantable rat Leydig cell tumor (H-540). Both isoproterenol and PGE1 caused activation of the AC in Leydig cell tumors. The degree of activation by PGE1 (4-5-fold) was approximately twice that of isoproterenol (2-3-fold). The addition of both AC agonists simultaneously was not additive indicating that they activate AC of the same cell. Increasing concentrations of ATP (0.025-2.0 X 10 mM) caused a concentration dependent increase in both the basal and hormone stimulated AC activity, and the activation by isoproterenol and PGE1 (relative response) revealed a slight but significant increase with increasing ATP concentrations. Lineweaver-Burke analysis of these data indicated an apparent Km for ATP (Mg X ATP) of 0.16 mM. Free magnesium did not influence the apparent Km of the AC for ATP. Increasing concentrations of free Mg2+ (0.24-13.2 mM) also caused a concentration dependent increasing activation of AC activity up to a concentration of approx 6 mM in excess of Mg2+-binding ingredients. Higher concentrations of free Mg2+ (13.1 mM) caused a small but significant decrease in both basal and agonist stimulated AC activity. In contrast to other reports, activation by isoproterenol and PGE1 was in general not influenced by the concentration of Mg2+. Both GTP and GMP-P(NH)P stimulated basal and hormone stimulated AC activity (Kact 1 microM), but with different kinetics. In the presence of GTP, AC activity was almost constant for 90 min. In the presence of GMP-P(NH)P, AC activity was much higher, but constant AC activity occurred after a certain lag time (7-10 min), which was reduced by PGE1 and isoproterenol. In conclusion, cAMP production in Leydig cell tumors is stimulated by both PGE1 and isoproterenol. The AC activity and activation by these agonists are regulated by Mg2+ and nucleotides in a slightly different manner from most other cells. The association between AC activation and stimulation of steroid production by Leydig cell tumors remains to be investigated.     

核苷对ROS17/2.8细胞株腺苷酸环化酶活性的影响

 大鼠成骨肉瘤细胞株(ROS17/2.8)系甲状旁腺素(PTH)的靶细胞。当该细胞质膜上的PTH受体与PTH结合后,可激发腺苷酸环化酶(AC)的活性。腺苷及四种不同的核苷酸(AMP、GMP、UMP、CMP)单独对AC无明显效应,但却可抑制PTH对AC的刺激作用。而鸟苷或木糖腺苷则可显著增强PTH对AC的刺激作用。提示核苷的不同代谢物在代谢调节中的多样化作用。     

Adenylate cyclase responses to sucrose stimulation in membranes of pig circumvallate taste papillae.

1. Typical adenylate cyclase (AC) responses to guanine nucleotides were found in membranes of pig circumvallate (CV) taste papillae. 2. Sucrose stimulated AC activity in the CV membranes and this stimulation was GTP dependent and tissue specific. 3. The stimulatory effect of sucrose in the CV membranes was dependent on the concentration of membranes used in the AC assay. 4. This study provides the first biochemical data on cellular transduction of taste in the pig, compares positively to preliminary results in cattle and supports recent suggestions for a role of cAMP in sweet taste transduction.     

Hormonal activation of adenylate cyclase in macrophage membranes is regulated by guanine nucleotides

Many macrophage functions such as chemotaxis, phagocytosis, enzyme secretion, and cytotoxicity are influenced by intracellular cyclic nucleotide levels, but the regulatory mechanisms involved are poorly defined. We have developed methods that allowed us to study the activation of AC in isolated guinea pig (g.p.) macrophage membranes. AC in these membrane preparations could be stimulated approximately twofold by guanine nucleotides. We could not obtain any hormonal activation of membrane-bound AC in the absence of guanine nucleotides. In the presence of GTP, however, the hormones isoproterenol and PGE1 elicited an additional threefold rise in AC activity, which subsided after approximately 15 min. As little as 10(-8) M concentrations of these two hormones induced significant elevations of AC activity. Replacement of GTP by its nonhydrolyzable analogue Gpp(NH)p resulted in a persistent hormone-independent activation of AC, and addition of hormones enhanced this level of activation. Thus, GTP-ase activity is present in macrophage membrane preparations and serves to regulate AC activation. Hormonal stimulation of AC was receptor mediated, because the effect of the beta-adrenergic agonist isoproterenol, but not PGE1, was inhibited by the beta-adrenergic blocker propranolol. In addition, the potency series of PG corresponded to that observed for stimulation of cAMP production in intact g.p. macrophages, i.e., PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. AC activation by PG in the membrane preparation was inhibited by an alpha-adrenergic agonist, thus demonstrating one means for down regulating cAMP production in g.p. macrophages. Our studies also showed that certain hormones (e.g., beta-adrenergic agonists, PG) can exert their effect on cAMP production by stimulation of membrane-bound AC, whereas other agents such as lectins or arachidonic acid require additional intracellular components to elevate cAMP levels in macrophages. The mechanism of activation of AC by hormones in g.p. macrophage membranes appears to fit the model of a ternary complex, the components of which include the hormone receptor, AC, and guanine nucleotide regulatory protein, which transmits the signal from the receptor to AC.     

Ethanol''s Effects on Cortical Adenylate Cyclase Activity

The effects of ethanol on beta-adrenergic receptor-coupled adenylate cyclase (AC) of mouse cerebral cortex were examined. The addition of ethanol (20-500 mM) to incubation mixtures containing cortical membranes demonstrated that ethanol could increase AC activity and potentiate the stimulatory effects of guanylyl-imidodiphosphate [Gpp(NH)p] on AC activity. Ethanol increased the rate of activation of AC by guanine nucleotides and concomitantly decreased the EC50 for magnesium required to achieve maximal stimulation of cortical AC. The EC50 values for Gpp(NH)p and isoproterenol stimulation of AC activity were also altered by ethanol. Ethanol was capable of stimulating AC extracted by use of digitonin. The AC activity in the digitonin extract was no longer sensitive to the addition of Gpp(NH)p or NaF, but was still stimulated by ethanol. We propose multiple sites of action for ethanol in stimulating cortical AC activity. These sites include actions at the beta-adrenergic receptor, at the G/F coupling proteins, and at the catalytic unit of cortical AC. Comparison of ethanol's actions on cortical beta receptor coupled AC activity with prior reported actions of ethanol on striatal dopamine (DA)-sensitive AC indicated differential sensitivities of these two AC systems to ethanol. These differences may be determined by specific coupling characteristics of the striatal and cortical AC systems or by differences in the plasma membranes in which striatal and cortical AC systems are located.     

牛脑皮层G_i的纯化及其与AC的偶联

用1％胆酸钠和20％饱和度的硫酸铵抽提牛脑皮层细胞膜得到含G蛋白和腺苷酸环化酶（AC）的制剂,通过Sepharose6B柱将两者分开,再将含G蛋白的级分用庚胺-Sepharose4B疏水柱、羟基磷灰石柱将其它亚型的G蛋白（主要是Gs和Go）从抑制型G蛋白（Gi）中除去,获得纯化的高活力的Gi,其GTP结合活力为17．6nmol／mg,比细胞膜Gi活力提高50倍;并具有较高的产率,从1g膜蛋白中可获得0．66mg的Gi,同时可获得无G蛋白污染的AC和少量的Gs蛋白．SDS-PAGE显示分子量为41000和36000的两条蛋白带,证实是Gi的α基和β亚基．进一步用重建脂酶体的方法检测Gi对AC的抑制作用,结果显示Gi对AC活力的抑制达40％左右,表明CAMP信息跨膜转导通路中Gi与AC之间具有较好偶联功能．     

MANT-substituted guanine nucleotides: a novel class of potent adenylyl cyclase inhibitors

Mammals express nine membranous adenylyl cyclase (AC) isoforms (AC1-AC9), but the precise functions of AC isoforms are still incompletely understood. This situation is at least partially due to the paucity of potent and isoenzyme-specific AC inhibitors. The original aim of our research was to develop a fluorescence assay for the stimulatory G-protein of AC, G(s). 2'(3')-O-(N-methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and were previously used for the fluorescence analysis of purified G(i)/G(o)-proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTPgammaS) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on Galpha(s)- and Galpha(i)-mediated signaling. MANT-GTPgammaS and MANT-GppNHp had lower affinities for Galpha(s) and Galpha(i) than GTPgammaS and GppNHp. In contrast to guanosine 5'-[beta-thio]diphosphate, MANT-GTPgammaS noncompetitively inhibited GTPgammaS-stimulated AC in Galpha(s)-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPgammaS and MANT-GppNHp was not due to Galpha(s) inhibition since it was also observed in Galpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked Galpha(i)-mediated AC inhibition by GTPgammaS and GppNHp in S49 cyc(-) membranes but not AC inhibition by MANT-GTPgammaS and MANT-GppNHp. MANT-GTPgammaS and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 nM and 160 nM, respectively. Taken together, MANT-substituted guanine nucleotides constitute a novel class of potent competitive AC inhibitors. The availability of potent fluorescent AC inhibitors will help us study the kinetics of AC/nucleotide interactions as well as function, trafficking and localization of AC isoenzymes in intact cells. In future studies, we will examine the specificity of MANT-nucleotides for AC isoenzymes.     

Plant purine nucleotide cyclases

Cyclic nucleotides (cAMP and cGMP) play an essential role in many important cellular processes in prokaryotic and eukaryotic organisms. They are produced by purine nucleotide cyclases: adenylyl and guanylyl cyclases. They are classified as one of two distinct forms: soluble and bound to membranes. Beside the differences in enzyme localization, the domain structure and regulation of enzymes activity are also diverse. However, all cyclases possess three groups of important residues: substrate specifying residue, metal binding residues and transition state stabilization residues. The natural occurrence of cyclic nucleotides in plants is now established. It was shown that in higher plants cNMPs act as a second messengers in a large number of (patho)physiological responses. However, it is only recently that the first plant enzymes with AC and GC activity of the unique structure have been identified and functionally characterized. In this study a systematic analysis of all the known prokaryotic, fungal and animal cyclases was done and direct evidences for the presence AC and GC in plant cells were shown.     

Nitric oxide-dependent allosteric inhibitory role of a second nucleotide binding site in soluble guanylyl cyclase

The mechanism of desensitization of the nitric oxide (NO) receptor (alpha1.beta1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of alpha1.beta1, based on the x-ray crystal structure of adenylyl cyclase (AC) suggest the existence of a nucleotide-like binding site, in addition to the putative catalytic site. We have previously reported that mutating residues that coordinate Mg(2+)GTP (substrate) binding in alpha1.beta1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of alpha1.beta1 (GC-wt) and the mutant form (AC-mut, alpha1R592Q.beta1E473K,C541D) were purified, and their sensitivities to various nucleotides were assessed. In using the AC-mut as well as other mutants that coordinate purine binding, we were able to distinguish allosteric inhibitory effects of guanine nucleotides from competitively inhibitory effects on catalytic activity. Here we report that several nucleotide analogs drastically alter sGC and AC-mut activity by acting at a second nucleotide site, likely pseudosymmetric to the catalytic site. In particular, Mg(2+)GTP gamma S and Mg(2+)ATP gamma S inhibited cyclase activity through a mixed, non-competitive mechanism that was only observable under NO stimulation and not under basal conditions. The non-competitive pattern of inhibition was not present in mutants carrying the substitution beta1D477A, the pseudosymmetric equivalent to alpha1D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations alpha1E525K,C594D, the pseudosymmetric equivalent to beta1E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.     

The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line,CRI-G1

The activity of a calcium-activated nonselective (Ca-NS⁺) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, -NAD⁺, -NADH, -NADP⁺ and -NADPH were very similar when tested at 0.1 mm, and at 1 mm the phosphorylated forms, -NADP⁺ and -NADPH, appeared to be slightly more potent than -NAD⁺ and -NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch.The application of -NAD⁺, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the -NAD⁺-NS⁺ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.     

Effect of organic ligands with conjugated π-bonds on the structure of iodine-α-dextrin complexes

Using X-ray data for iodine-α-dextrin complexes and the results of quantum chemical ab initio restricted Hartree-Fock/3-21G(**) level calculations, a model of drug active complex (AC) Armenicum with anti-HIV action was proposed. It was suggested that the drug AC contains molecular iodine allocated inside of α-dextrin helix and coordinated by lithium halogenides and a protein component of lymphocyte ribosomes. The electronic structure of I(2) in this complex differs from its characteristics in complexes with organic ligands or the free I(2) . In the considered ACs, the molecular iodine displays acceptor (donor) properties toward the α-dextrins (lithium halogenides). A mechanism of Armenicum anti-HIV action is suggested. Under the influence of molecular iodine-containing drug AC, the structure of HIV DNA is modified-it becomes more π-donor-active against proteins and peptide nucleotides of viral DNA form a stable complex with molecular iodine and lithium halogenides.     

Molecular Characterization of a Strain of Squash leaf curl China Virus from North India

A Begomovirus causing yellow vein mosaic disease of pumpkin (Cucurbita maxima L.) was characterized at molecular level by cloning and sequence analysis of its complete DNA‐A genome. The DNA‐A of the isolate contains 2758 nucleotides which encode six open reading frames (ORFs): AV1 and AV2 in the virion‐sense and AC1, AC2, AC3 and AC4 in the complementary‐sense. Based on the highest (96%) sequence identities and close phylogenetic relationships with Squash leaf curl China virus species, the Begomovirus was identified as strain of Squash leaf curl China virus. The presence of DNA‐B genome of the virus strain was also detected by dot blot hybridization test using DNA‐B specific probe.     

Regulation of the Adenylyl Cyclase System of the Infusorian Tetrahymena pyriformis by Hormonal and Non-Hormonal Agents and Its Dependence on the Basal Activity Level of Adenylyl Cyclase

Effects of glucagon and biogenic amines as well as of non-hormonal agents (manganese cations, forskolin, guanine nucleotides) on activity of adenylyl cyclase (AC) was studied in the infusorian Tetrahymena pyriformis, a representative of unicellular eukaryotes. The infusorian cultures at different growth stages were chosen for the study. It has been revealed that the AC basal activity measured in the cultures at the stationary stage is much lower than in those at the exponential stage. The stimulating effects of hormones (glucagon, serotonin, isoproterenol) and of non-hormonal agents were shown to depend markedly on the tetrahymena AC basal activity. These effects are pronounced at the stationary stage of growth (the low basal activity), whereas in cultures at the active division stage (the high basal activity) they either are reduced or change their sign to opposite (inhibition of AC activity. The obtained results not only are another evidence in favor of the existence of the functionally active ACS in lower eukaryotes, but also indicate an important role of this system in regulation of sensitivity of infusoria at different growth stages to external actions.     

AC133-2, a novel isoform of human AC133 stem cell antigen

Human AC133 antigen, also called CD133, was recently identified as a hematopoietic stem cell marker. However, the molecular structure and function of this protein has remained unclear. Here we cloned and identified a novel isoform of AC133, which we named AC133-2. In comparison to the reported AC133 cDNA, which is referred to herein as AC133-1, a small exon of 27 nucleotides is deleted in AC133-2 by alternative mRNA splicing. Similar to the previously characterized AC133 antigen, recombinant AC133-2 expressed in 293 cells was glycosylated and transported to plasma membrane. AC133-2 mRNA was found predominant in a variety of human fetal tissue, adult tissues, and several carcinomas. In contrast, AC133-1 mRNA was more prominent in fetal brain and adult skeletal muscle but was not detected in fetal liver and kidney, adult pancreas, kidney, and placenta, suggesting different roles for the two isoforms in fetal development and mature organ homeostasis. Here, we demonstrate that AC133-2 is the isoform expressed on hematopoietic stem cells derived from fetal liver, bone marrow, and peripheral blood. The results indicate that AC133-2, not AC133-1, has been the cell surface antigen recognized by anti-AC133 monoclonal antibodies that are used for isolation of hematopoietic stem cells. To further investigate its expression in other stem cell populations, we found that AC133-2 co-expressed with beta(1) integrin in the basal layer of human neonatal epidermis. AC133-2(+)/beta(1) integrin(+) cells proliferated and differentiated in culture, which coincided with a loss of AC133-2 and gain in a terminal differentiation marker involucrin. Taken together, these results suggest that AC133-2 is expressed in multiple stem cell niches and may provide a means to isolate specific stem cell subpopulations from human tissues.     

广东番茄曲叶病毒G2分离物基因组DNA-A的分子特征

从采集于广东的番茄曲叶病病株上分离到病毒分离物G2 ,序列分析结果表明 ,其DNA_A为单链环状 ,全长2 74 4nt,共有 6个ORF ,其中病毒链上编码AV1(CP)、AV2 ,互补链上编码AC1、AC2、AC3和AC4。BLAST结果显示 ,与G2基因组有同源关系的病毒均属双生病毒科菜豆金色花叶病毒属。序列比较结果显示 ,G2与菜豆金色花叶病毒属病毒的DNA_A序列同源率均不超过 83% ,其中同源率最高的是PaLCuCNV_[G10 ](82 8% )。进一步比较发现 ,它们的基因间隔区 (IR)变异最大 (同源率为 30 9%～ 81 8% ) ;CP氨基酸序列的同源率较高 (77 6 %～ 99 2 % ) ,AC4蛋白氨基酸序列的同源率较低 (4 3 5 %～ 78 8% )。系统进化关系分析结果也显示 ,G2与已报道的菜豆金色花叶病毒属病毒的亲缘关系均较远。因此 ,G2可能是双生病毒科菜豆金色花叶病毒属中一个未报道的新种 ,命名为广东番茄曲叶病毒 (TomatoleafcurlGuangdongVirus ,ToLCGDV)     

2'-'5-三腺苷酸对巨噬细胞腺苷酸环化酶和cAMP-磷酸二酯酶活性的影响

1989年,我们曾首次证实干扰素作用介导物pppA2＇p5＇A2＇p5＇A(2＇-5＇-三腺苷酸,2＇-5＇P_3A_3)能引起巨噬细胞中cAMP,cGMP水平升高,表明这两种环核苷酸在传递干扰素信息中起着重要作用。在上述研究的基础上,本研究观察了2＇-5＇P_3A_3对腺苷酸环化酶(AC)和cAMP-磷酸二酯酶(cAMP-PDE)两种酶的活性影响,结果发现,1×10~(-6)mol/L的2＇-5＇P_3A_3可显著增加AC的活性,而对cAMP-PDE活性沒有显著影响,这说明2＇-5＇P_3A_3引起的细胞内cAMP水平的升高是由于激活AC而使其生成增多,而不是抑制cAMP-PDE而使其降解减少的结果。