腺苷三磷酸和环腺苷单磷酸对丝状真菌纤维素酶合成的调节

以虫荧光素酶法检验了四株丝状真菌在葡萄糖—无机盐液体培养过程中的胞内ATP含量。结果表明,只有当胞内ATP浓度低于10~(-S)mg/ml时,真菌才开始合成胞外纤维素酶(FPA)。以不同浓度的各种碳源培养时,菌体胞内ATP含量只要超过10~(-1)mg/ml,FPA的合成即发生阻遏。菌体胞内ATP含量与FPA合成呈显著负相关。以高效液相色谱(HPLC)法检测了菌体培养液中的cAMP含量。在非阻遏条件下,外源cAMP可以提高FPA的合成水平。但外源cAMP不能解除已经发生的酶合成阻遏。菌体ATP和cAMP水平是调节真菌纤维素酶合成的重要因子。     

Measurement of cyclic 3',5'-adenosine monophosphate synthesis in growing Escherichia coli.

The net synthesis of cAMP by an adenine auxotroph of $\text{Escherichia coli}$ was measured by assaying the incorporation of tritium from [³H]-adenine into cyclic [³H] AMP during exponential growth. Synthesis of cAMP ceased abruptly when glucose was added to cells growing in glycerol and then recovered to an intermediate rate of synthesis after 0.5–1.0 generation. Cyclic AMP appeared to be synthesized from a precursor pool that turned over more rapidly than total cellular ATP. The rates of cAMP synthesis measured by this technique are compatible with the cellular levels of cAMP previously measured in this strain(3).     

cAMP dependent inhibition of thromboxane A2, prostacyclin and PGF2α synthesis in mouse hepatocytes

The role of cAMP dependent regulation in thromboxane A₂, prostacyclin and PGF_2α synthesis (measured by radioimmunoassay) was investigated in isolated mouse hepatocytes and in microsomal membranes prepared from these cells. In isolated hepatocytes N⁶,O²-dibutyryl cAMP inhibited the formation of all the three derivatives, while calcium ionophore A 23187 stimulated their synthesis. Addition of the dissociated catalytic subunit of cAMP dependent protein kinase and ATP to microsomal membranes inhibited the production of TXA₂, PGI₂ and PGF_2α by about 50% and this inhibition was counteracted by the combined addition of heat stable inhibitor protein of cAMP dependent protein kinase. It is concluded that in parenchylmal liver cells cAMP dependent phosphorylation is directly involved in the inhibition of prostanoid synthesis.     

Induction of cellulase in trichoderma reesei grown on lactose

Intracellular concentrations of ATP, cyclic AMP and glucose-6-phosphate were monitored during growth of partially catabolically derepressed strain of Trichoderma reesei CC II in medium containing lactose as the sole carbon source. The induction of cellulase synthesis occured when the concentration of lactose in the medium decreased below 7 mg/ml. The onset of cellulase synthesis was preceded by a transient peak of intracellular concentration of ATP and by the increase of the cyclic AMP contents in the mycelium whereby the intracellular level of glucose-6-phosphate was at its minimum. By keeping the lactose concentration in the medium at 2 mg/ml, it was possible to support the continuation of cellulase synthesis over the prolonged periods without appreciable growth of biomass.     

Cyclic Adenosine Monophosphate in the Nervous System of Aplysia californica : I. Increased synthesis in response to synaptic stimulation

In the isolated abdominal ganglion of Aplysia, previously incubated in adenine-³H, the amount of ³H-labeled adenosine-3',5' monophosphate (cAMP) doubled after electrical stimulation of nerves at a physiological rate (1/sec). No change was detected after 4 min of stimulation. An increase in cAMP was first seen after 15 min; lengthening the period of stimulation to 1 hr did not increase the extent of the effect. ATP contained 50% of the total radioactivity taken up from adenine-³H, cAMP about 0.1%. During stimulation both the total amount and the specific radioactivity of adenosine triphosphate (ATP) did not change. Thus, the increased amount of radioactivity found in cAMP after stimulation represented an increase in its rate of synthesis. During stimulation formation of cAMP-³H was not altered in nerves or in the cell body of an identified neuron (R2). In addition, no changes were detected in the total amounts of cAMP in the ganglion and in the cell body of R2. It seems likely that the increase was initiated by synaptic activity rather than by action potentials. It was blocked by elevating the concentration of Mg, which also blocks synaptic activity without impairing conduction of impulses. Moreover, impulse activity induced by ouabain and glutamate did not result in increased formation of cAMP.     

Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many ³²P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-³²P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-³²P] phosphate from [γ-³²P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]₀ e^?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [³²P] phosphate from [γ-³²P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.     

Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound,cAMP-activated protein kinase

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3′,5′-cyclic monophosphate (8-N₃ cAMP), and [γ³²P] ATP, the membranebinding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [³²P] 8-N₃ cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [γ³²P] ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg · ATP concentration is below approximately 10 μM. The catalytic subunit is not required for the Mg · ATP inhibition of 8-N₃ cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N₃ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N₃ cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied in other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg · ATP.     

Characterization of Cyclic AMP Efflux from Swine Adipocytes In Vitro

Objective : A variety of cell types transport cyclic AMP (cAMP) to the extracellular fluid; the purpose of this study was to determine if and how this process occurs in adipocytes. Research Methods and Procedures : Adipocytes were isolated from 3-month-old swine and incubated with stimulators of adenylate cyclase for 2 to 120 minutes to promote cAMP synthesis and efflux. Efflux was characterized in the presence of agents that inhibit ATP production, anion transport, intracellular cAMP metabolism, and extracellular cAMP metabolism. Extracellular cAMP was measured by enzyme immunoassay, then corrected for cell lysis by measuring lactate dehydrogenase release. Results : cAMP efflux averaged 24.7 fmol/min/cm² adipocyte surface area, was linear for 2 hours, and was proportional to adipocyte surface area (r = 0.94, p<0.05). Efflux was reduced by ∽35% in cells incubated with 1 4mUM antimycin, an inhibitor of ATP synthesis (p<0.05), and by ～55% in cells incubated with 2 mM probenecid, an anionpecific transport blocker (p<0.05). Extracellular cAMP levels more than doubled by the addition of 1 μM 1,3-dipropyl-8-p-sulfophenylxanthine, a purported inhibitor of extracellular phosphodiesterase. Discussion : Our data demonstrate that cAMP is transported from swine adipocytes by an energy-dependent anion transporter and can be metabolized extracellularly. Future studies will evaluate extracellular cAMP as a potential source of extracellular adenosine, a potent inhibitor of adipocyte lipolysis.     

An ATP pool with rapid turnover within the cell membrane

Seven-day-old cultures of rat leg muscle cells were double labelled by addition of [¹⁴C] adenine in the culture medium (2 hrs 15 mins) and followed by addition of [³²P] phosphate (15 min). The specific activity (S.A.) of the isolated cyclic [¹⁴C] adenine 3′ – 5′ monophosphate (cAMP) was similar to that of the bulk ATP. The S.A. of [³²P] from cAMP was, however, higher than that of bulk ATP. The S.A. of [³²P] from cAMP could be further modified by prevention of normal muscle cell fusion. It is probable that the cAMP with high [³²P] S.A. was synthesized from a cell membrane pool of ATP with rapid turnover.     

Different mechanisms of hydroxyl radical production susceptible to purine P2 receptor antagonists between carbon monoxide poisoning and exogenous ATP in rat striatum

Abstract

Previous studies have suggested that carbon monoxide (CO) poisoning stimulates cAMP production via purine P2Y11-like receptors in the rat striatum, activating cAMP signaling pathways, resulting in hydroxyl radical (^?OH) production. Extracellular ATP was thought likely to trigger the cascade, but the present study has failed to demonstrate a clear increase in the extracellular ATP due to CO poisoning. The CO-induced ^?OH production was attenuated by the P2Y11 receptor antagonist NF157, in parallel with its abilities to suppress the CO-induced cAMP production. The ^?OH production was more strongly suppressed by a non-selective P2 receptor antagonist, PPADS, which had no effect on cAMP production. More selective antagonists toward the respective P2 receptors susceptible to PPADS, including NF279, had little or no effect on the CO-induced ^?OH production. The intrastriatal administration of exogenous ATP dose-dependently stimulated ^?OH production, which was dose-dependently antagonized by PPADS and NF279 but not by NF157. Exogenous GTP and CTP dose-dependently stimulated ^?OH production, though less potently. The GTP-induced ^?OH production was susceptible to both of NF279 and PPADS, but the CTP-induced ^?OH production was resistant to PPADS. The mechanism of ^?OH production may differ between CO poisoning and exogenous ATP, while multiple P2 receptors could participate in ^?OH production. The CO-induced ^?OH production was susceptible to the inhibition of NADPH oxidase, but not xanthine oxidase. Also, the NADPH oxidase inhibition suppressed ^?OH production induced by forskolin, a stimulator of intracellular cAMP formation. It is likely that ^?OH is produced by NADPH oxidase activation via cAMP signaling pathways during CO poisoning.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 μM-1 mM and was induced at different values of intracellular pH and Ca²⁺. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg²⁺ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca²⁺ and their sensitivity to intracellular cAMP are consistent with a role in the Ca²⁺ oscillations associated with cell growth.     

Method for measuring 32P-labeled cAMP levels in intact tissue.

A new method has been developed for prelabeling tissue ATP pools with ³²P inorganic phosphate (P_i) and for the subsequent isolation of [³²P]cAMP and [³²P]ATP. The new method of prelabeling eliminates the need to separate trace amounts of radioactive cAMP from radioactive breakdown products of adenine formed in tissues prelabeled with [³H]- or [¹⁴C]adenine. The effect of epinephrine to increase [³²P]cAMP levels in rat ventral prostate tissue fragments has been studied in terms of increase in the ratio of [³²P]cAMP/[³²P]ATP in the absence and presence of various phosphodiesterase inhibitors. Tissue prelabeling with ³²P_i labels GTP as well as ATP (and other nucleoside triphosphates); thus the method lends itself to the isolation of [³²P]cGMP as well as [³²P]cAMP from the same tissue sample.     

Hormonal regulation of acetyl CoA carboxylase effect of insulin and epinephrine

Acetyl CoA carboxylase in the isolated epididymal fat tissue is activated by insulin and inactivated by epinephrine and dibutyryl cAMP. Insulin activation of the enzyme occurs in the presence of inhibitors of protein synthesis and can be observed as early as 15 min. A time dependent inactivation of the enzyme occurs in the presence of ATP and Mg⁺⁺$\text{in vitro}$.     

cAMP/Protein Kinase A Activates Cystic Fibrosis Transmembrane Conductance Regulator for ATP Release from Rat Skeletal Muscle during Low pH or Contractions

We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTR_inh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTR_inh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na⁺/H⁺-exchanger (NHE) with amiloride or the Na⁺/Ca²⁺-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway.     

Adsorption chromatography of cyclic nucleotides on silica gel and alumina thin-layer sheets

Several solvent combinations of graded polarity have been developed, capable of separating cAMP or cGMP, not only from related nucleotides but from bases and nucleosides on alumina and silica gel thin-layer sheets (tls). The solvent system chloroform (C), methanol (M), water (W) (40:20:3) gave effective separations of cAMP on silica gel TLS. Identical results were obtained when this adsorption chromatography method was compared with the ion-exchange chromatography method involving PEI for its effectiveness in the separation of [α-³²P]cAMP formed from [α-³²P]ATP by a preparation of bovine sperm cells. In addition, this solvent system effectively separates cAMP from inosine, hypoxanthine, xanthine, and uric acid which may be useful in determinations of cAMP arising from ³H- or ¹⁴C-prelabeled ATP. Effective separation of cGMP on silica gel tls was accomplished with C:M:W (30:30:5). On alumina tls, cAMP and cGMP were separated from related compounds with M:W combinations; the solvent M:W (50:50) gave the lowest blanks (0.02%) for cGMP and it may prove useful in cGMP determinations.     

Studies on the inhibition a fatty acid synthesis in the chicken liver by adenine compoundsin vitro

The biosynthesis of fatty acids from [l-¹⁴C]-acetate in the chicken liver slicesin vitro was inhibited by cAMP, adenosine, 5′-AMP, 3′-AMP, ATP, NAD and FAD but not by adenine, guanine or inosine. The minimum structural requirement for inhibition appears to be adenosine. The inhibitory action of adenosine, 5′-AMP and NAD on fatty acid synthesis is likely to be mediated by adenosine or its metabolites since adenosine deaminase reverses the inhibition while it has no effect on the inhibition by cAMP; thus, the inhibitory effect of cAMP is probably not mediated through its hydrolysis products, 5′-AMP, or adenosine.     

Homologous desensitization of ATP-stimulated mitogenesis: Mechanism involves desensitization of arachidonic acid release and cAMP elevation but not the activation of protein kinase A

Prolonged incubation of quiescent 3T3, 3T6, and A431 cells with the P_2Y purinoceptor agonists ATP, ADP, or AMPPNP reduced the mitogenic responses of target cells to a further challenge by these agonists, as measured by [³H]thymidine incorporation. The mitogenic desensitization was agonist-specific, for no effect was seen on DNA synthesis stimulated by epidermal growth factor, insulin, bombesin, 12-0-tetradecanoyl-phorbol-12 acetate (TPA), or adenosine. The desensitization was completely reversible, since after a 24 hr incubation in the absence of ATP, the cells responded fully to the mitogenic action of ATP. The presence of a low level of cycloheximide blocked recovery, suggesting that down-regulation of the P_2Y receptor may have occurred during desensitization. In Swiss 3T3 cells, stimulation of DNA synthesis occurs predominantly by activation of arachidonic acid release, followed by its oxidation to prostaglandin E₂ and stimulation of adenylyl cyclase. Interestingly, prolonged preincubation with ATP produced a similar degree of desensitization of DNA synthesis and of ATP-dependent arachidonic acid release and cAMP accumulation. Furthermore, this was true for both wild type cells and mutants with a defective cAMP-dependent protein kinase (PKA). We conclude that homologous desensitization is likely due to uncoupling of the P_2Y purinoceptor from phospholipase A₂, and this process does not require activation of protein kinase A. © 1995 Wiley-Liss Inc.     

产碱性纤维素酶菌株的选育和酶合成基本特性

芽孢杆菌x－6菌株经甲基磺酸乙酯（EMS）和紫外线（UV）复会诱变,从其万古霉素（Vm）抗性突变体中选育获得一突变株EV23,所产生的碱性核甲基纤维素酶（CMCase）酶活力由原来的0．84u／ml提高到3.53u／ml。EV23菌株所产该酶基本为组成性地合成纤维素酶,酶合成明显表现出抗降解物阻遏的特点,以葡萄糖为碳源培养,4％浓度时酶合成水平最高。酶合成效率受菌体生长速率影响较大。在高浓度易代谢基质和三羧酸循环中间物存在下,酶合成将受到一定程度的阻遏。酶合成还与能量代谢有关,探讨了外源ATP、cAMP对     

An Improved, Automated Adenylate Cyclase Assay Utilizing Preparative HPLC: Effects of Phosphodiesterase Inhibitors

Abstract: Analysis of adenylate cyclase (ACase) activity in broken cell preparations usually involves conversion of [α-³²P]ATP to [³²P]cyclic AMP (cAMP) followed by purification of cAMP by liquid chromatographic methods. An automated, preparative reverse-phase HPLC procedure was developed that purifies cAMP rapidly and decreases variability and background. It permits the separation procedure to be validated rapidly prior to use with actual samples, and is readily adaptable for assaying guanylate cyclase, phosphodiesterases (PDE), or a variety of other related nucleotide-metabolizing enzymes. For ACase assays, 4.5% ZnSO₄-10% Ba(OH)₂ is added to the incubation mixture, and following centrifugation, the supernatant is injected on an HPLC apparatus fitted with a Waters Z-Module containing a 10-μ C₁₈ reverse-phase cartridge. Using a mobile phase of 0.15 M sodium acetate-20% methanol (pH 5.0) at a flow rate of 4 ml/min, cAMP is eluted at k′ > 1.25, whereas k′ < 0.5 for all other adenine nucleotides, permitting collection of the cAMP fraction after running the other nucleotides to waste. The method was validated by characterizing dopamine-sensitive ACase in homogenates of striatum from Sprague-Dawley rats. Basal activity (177 ± 16 pmol/mg protein/min), the stimulation by dopamine (186 ± 19 pmol/mg/min), the apparent K_m for dopamine (5.0 ± 1.5 μM), and expected effects of varying magnesium, EGTA, and GTP were similar to available data. However, it was found that isobutylmethylxanthine (IBMX) or theophylline, usually included in the incubation mixture as PDE inhibitors, markedly inhibited the synthesis of cAMP in both the presence and absence of dopamine. A consequence of this inhibition was a marked change in the apparent K_m of dopamine calculated from a Lineweaver-Burk plot. The use of IBMX to inhibit PDEs was compared with an alternate strategy, the addition of excess exogenous cAMP. Simultaneous analysis of PDE and ACase activity was accomplished by including [³H]cAMP in the incubation and quantifying the amounts of [³H]cAMP hydrolyzed and [³²P]cAMP synthesized. Without IBMX, a concentration of 1 mM exogenous cAMP was sufficient to prevent significant loss of [³H]cAMP. In the absence of exogenous cAMP, 0.5 mM IBMX did not completely prevent the breakdown of [³H]cAMP, whereas 2.5 mM IBMX did. Although there was 25% less [³H]cAMP recovered in the presence of 0.5 mM IBMX than with 2.5 mM IBMX, there was no difference in the amount of [³²P]cAMP formed (either with or without dopamine). Moreover, in the presence of IBMX, there was a 20–30% lower synthesis of [³²P]cAMP compared with incubations in which only 1 mM cAMP was used to prevent breakdown of [³²P]cAMP. These data suggest that alkylxanthines, possibly through effects on adenosine receptors, may cause unexpected effects on estimations of dopamine-stimulated ACase. The use of exogenous cAMP as an alternate substrate for PDEs may be one way to obviate these problems.