共查询到20条相似文献,搜索用时 15 毫秒
1.
Spangler CM Spangler C Göttle M Shen Y Tang WJ Seifert R Schäferling M 《Analytical biochemistry》2008,381(1):86-93
Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-32P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (KM), the v0max value of ATP turnover, and the IC50 values for three inhibitors of EF by this newly developed fluorescent method. 相似文献
2.
N C Sangster D F Mettrick 《Comparative biochemistry and physiology. B, Comparative biochemistry》1987,88(1):317-321
1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK activity. 相似文献
3.
Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM. 相似文献
4.
Jonathan H. Zippin Yanqiu Chen Susanne G. Straub Kenneth C. Hess Ana Diaz Dana Lee Patrick Tso George G. Holz Geoffrey W. G. Sharp Lonny R. Levin Jochen Buck 《The Journal of biological chemistry》2013,288(46):33283-33291
The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. 相似文献
5.
Hyun-Woo Rhee Kyoung-Shim Kim Pyung-Lim Han Jong-In Hong 《Bioorganic & medicinal chemistry letters》2010,20(3):1145-1147
In cellular signaling, adenylyl cyclase plays a key role in the hydrolysis of ATP to cyclic AMP and pyrophosphate. Using a synthetic fluorescent chemosensor (PyDPA) which binds strongly to the pyrophosphate group, we have developed a label-free fluorescent real-time detection system for adenylyl cyclase. This assay would be the first adenylyl cyclase assay based on chemosensing the production of pyrophosphate. 相似文献
6.
O. Bârzu F. Eckstein S. Dancea I. Petrescu C. Tărmure L.D. Ngoc A. Hodârnău H.H. Mantsch 《BBA》1979,547(2):361-369
Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria. 相似文献
7.
Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism 总被引:16,自引:0,他引:16
The Ca2+(calmodulin (CaM))-dependent protein kinase II, purified from either rabbit liver or rat brain, was preincubated under conditions that are known to promote its autophosphorylation. When kinase activity was assayed after this preincubation, it was observed that excess EGTA could block no more than 40-60% of the total Ca2+- and CaM-dependent activity compared to 95% inhibition by EGTA prior to preincubation. In the EGTA assay, free Ca2+ was calculated to be less than 1 nM; therefore, this activity was designated Ca2+-independent activity. Formation of this Ca2+-independent form of the kinase was shown to be associated with autophosphorylation based on the following observations: (a) it required the presence of Ca2+, CaM, and ATP; (b) the ATP analogs adenylyl imidodiphosphate and adenylyl methylenediphosphate could not substitute for ATP; (c) generation of the independent form was associated with incorporation of phosphate into the kinase; and (d) addition of protein phosphatase partially dephosphorylated the kinase and restored its Ca2+ dependence. This phenomenon may be of physiological importance because it would prolong the effects of extracellular signals that only transiently increase the intracellular Ca2+ level. 相似文献
8.
Roger A. Johnson Ilana Shoshani Carmen Dessauer Gilles Gosselin 《Nucleosides, nucleotides & nucleic acids》2013,32(4-5):839-842
Abstract An enzymatic method was developed for the preparation of unlabeled and [β-32P]-labeled β-L-2′,3′-dd-5′ATP from the monophosphate with near quantitative yields. β-L-2′,3′-dd-5′ATP was a competitive and potent inhibitor of adenylyl cyclases (IC5 ~ 30 nM). Upon uvirradiation β-L-2′,3′-dd-[β-32P]-5′ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2′,5′-dd-3′ATP, a post-transition state, noncompetitive inhibitor. 相似文献
9.
The manufacturing processes of many electronic and medical products demand the use of high-quality water. Hence the water supply systems for these processes are required to be examined regularly for the presence of microorganisms and microbial biofilms. Among commonly used bacteria detection approaches, the ATP luminescence assay is a rapid, sensitive, and easy to perform method. The aim of this study is to investigate whether ATP regeneration from inorganic pyrophosphate, a product of the ATP luminescence assay, can stabilize the bioluminescence signals in ATP detection. ADPglc pyrophosphorylase (AGPPase), which catalyzes the synthesis of ATP from PPi in the presence of ADPglc, was selected because the system yields much lower luminescence background than the commercially available ATP sulfurylase/adenosine 5′-phosphosulfate (APS) system which was broadly used in pyrosequencing technology. The AGPPase-based assay could be used to measure both PPi and ATP quantitatively and shows 1.5- to 4.0-fold slight increases in a 10-min assay. The method could also be used to stabilize the luminescence signals in detection of Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus in either broth or biofilm. These findings suggest that the AGPPase-based ATP regeneration system will find many practical applications such as detection of bacterial biofilm in water pipelines. 相似文献
10.
Philip G. Hoffman Oren Zinder Olga Nikodijevic Harvey B. Pollard 《Journal of cellular biochemistry》1976,4(2):181-184
ATP stimulates chromaffin granules from the bovine adrenal medulla to release epinephrine and specific soluble proteins. ATP analogs substituted in the β-γ-position with either nitrogen or carbon were also found to be effective at inducing release from isolated chromaffin granules. However, an ATP analog substituted at the α-β position with carbon was strongly inhibitory. Cyclic AMP was also found to be synthesized by isolated chromaffin granules under release conditions. ATP analogs were effective as substrates for adenylate cyclase in the same order as their efficiency for inducing release from vesicles. Hydrolysis at the β-γ linkage of ATP therefore is probably not necessary for release; however, hydrolysis at the α-β position may be important in the release process. Cyclic AMP may be produced and play a regulatory role in this event. 相似文献
11.
Transport of arginine and aspartic Acid into isolated barley mesophyll vacuoles 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The transport of arginine into isolated barley (Hordeum vulgare L.) mesophyll vacuoles was investigated. In the absence of ATP, arginine uptake was saturable with a Km of 0.3 to 0.4 millimolar. Positively charged amino acids inhibited arginine uptake, lysine being most potent with a Ki of 1.2 millimolar. In the presence of free ATP, but not of its Mg-complex, uptake of arginine was drastically enhanced and a linear function of its concentration up to 16 millimolar. The nonhydrolyzable adenylyl imidodiphosphate, but no other nucleotide tested, could substitute for ATP. Therefore, it is suggested that this process does not require energy and does not involve the tonoplast ATPase. The ATP-dependent arginine uptake was strongly inhibited by p-chloromercuriphenylsulfonic acid. Furthermore, hydrophobic amino acids were inhibitory (I50 phenylalanine 1 millimolar). Similar characteristics were observed for the uptake of aspartic acid. However, rates of ATP-stimulated aspartic acid transport were 10-fold lower as compared to arginine transport. Uptake of aspartate in the absence of ATP was negligible. 相似文献
12.
《Bioorganic & medicinal chemistry letters》2020,30(2):126823
Adenylyl cyclases (ACs), which are responsible for catalyzing the conversion of adenosine triphosphate (ATP) into the second messenger cyclic adenosine monophosphate (cAMP), play a critical role in cell signal transduction. In this study, a combined approach involving docking-based virtual screening, with the combination of homology modeling followed by an in-vitro, and cell-based biological assay have been performed for discovering a class of novel potent and selective isoform adenylyl cyclase type 8 (AC8) agonist. The computer-aided virtual screening was used to identify fourteen virtual cluster compounds as potential hits which were further subjected to rigorous bioassays. A novel hit compound VHC-7 (ethyl 3-(2,4-dichlorobenzyl)-2-oxoindoline-3-carboxylate) was identified as a highly potent selective AC8 agonist with EC50 value of 0.1052 ± 0.038 µM. Remarkably, the molecule herein reported can be explored further to discover greater number of hit compounds with better pharmacokinetic properties as well as to serve as a promising novel hit agonist of AC8 for the treatment of various central nervous system disorders and its associated diseases. 相似文献
13.
《Comparative biochemistry and physiology. C: Comparative pharmacology》1989,92(1):111-114
1. The actions of adenylyl compounds were investigated in the circular muscle of the pedal disc of the sea anenome Actinia equina.2. Adenosine, adenosine 5'-diphosphate and adenosine 5'-triphosphate (ATP), but not adenosine 5'-monophosphate or the analogues of ATP, α,β-methylene ATP, and 2-methylthio ATP, caused concentration-dependent contractions.3. Neurogenic contractions in response to electrical field stimulation were not consistently affected by any of the adenylyl compounds and could be either potentiated or almost abolished by them.4. Reactive Blue 2, a vertebrate P2-purinoceptor antagonist, caused concentration-dependent contractions which were mediated by nerves, being blocked by the anaesthetic chlorobutanol.5. The pharmacological profile of the adenylyl compounds in the pedal disc of Actinia equina is different from that observed in other invertebrate species and adds to the greater diversity of such profiles in invertebrates than in vertebrates. 相似文献
14.
《Journal of receptor and signal transduction research》2013,33(1-4):69-78
AbstractAn in situ assay able to detect hormonally stimulated or inhibited adenylyl cyclase (AC) activity on as few as 5,000 cells/well has been developed. In addition the assay monitors phosphatase activity which serves as a marker for cell density. Cells are plated in replicate wells at least one day before the assay, and the medium containing AC reagents, an ATP regenerating system, a PDE inhibitor, additives that regulate receptors and/or G proteins, and 5 mM pnitrophenyl phosphate (pNPP), plus Tris buffer to pH 7.5 is added. The hypotonic medium causes permeabilization of cells without massive lysis. After stopping the reaction with 100 μl of a solution with ATP, cAMP and SDS, the color indicating phosphatase activity is quantified by an ELISA reader, and AC activity measured by standard methods. At proper cell density (pNPP hydrolysis) the assay shows proportionality up to 2 hours. The assay is particularly useful in transfection experiments. As few as 50,000 cells can be plated and identified as receptor “positive” or receptor “negative”. The assay was key to our cloning of the V2 AVP receptor. The assay accelerated the preparation of stable cell lines with LH, FSH, adrenergic and serotonin IDβ/1B and IE receptors. It should also be useful in studies in which the transfected cDNA encodes the adenylyl cyclase proper. 相似文献
15.
Scott R. Whittemore† Stephen G. Graber‡ Robert H. Lenox Edith D. Hendley† Yigal H. Ehrlich‡ 《Journal of neurochemistry》1984,42(6):1685-1696
Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl2 resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation-dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg2+ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral-cortical membranes with adenosine-5′-0-(3-thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine-5′-0-(3-thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl2 also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of 35S from [35S]ATPγS into a protein component with apparent Mr of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATPγS. Endogenous activity utilizing [γ-32P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane-bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent Mr of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP-preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation. 相似文献
16.
Matthew L. Fowler Cheryl J. Ingram-Smith Kerry S. Smith 《Journal of visualized experiments : JoVE》2011,(58)
Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi. 相似文献
17.
DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme 总被引:9,自引:0,他引:9
We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound. 相似文献
18.
Thomas Emmrich Ali El-Tayeb Hesham Taha Roland Seifert Christa E. Müller Andreas Link 《Bioorganic & medicinal chemistry letters》2010,20(1):232-235
(2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methylamino)benzamido]tetrahydrofuran-2-yl-methoxy[(hydroxy)phosphoryloxy][(hydroxy)phosphoryl]dichloromethylphosphonic acid was synthesized as a chemically and metabolically stable analog of ATP substituted with a fluorescent methylanthranoyl (MANT) residue. The compound is intended for studying the binding site and function of adenylyl cyclases (ACs), which was exemplified by studying its interaction with Bacillus anthracis edema factor (EF) AC exotoxin. 相似文献
19.
Michel Fèvre 《Planta》1983,159(2):130-135
Membrane-bound β-1-3- and β-1-4-glucan synthetases of Saprolegnia are affected in vitro by the presence of nucleotides. Both enzymatic activities are inhibited by uridine nucleotides. Guanosine 5′-triphosphate and ATP reduce β-1-3-glucan synthesis but stimulate β-1-4-glucan production; they also increase V max without effect on the K m for uridine 5′-diphosphate glucose. The stimulation by ATP could be the result of an activation or stabilization of the enzymes and might have implications for cell-wall construction during hyphal growth. 相似文献
20.
Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1+B220+, a recently identified potent interferon (IFN)-γ producer. Indeed, IFN-γ was produced in those cultures, and pre-B cells lacking IFN-γ receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking β2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-γ beyond the selection imposed upon self-recognition. 相似文献