辅助能量物质强化环磷酸腺苷发酵合成机制 *

微生物代谢过程中,环磷酸腺苷(cAMP)由ATP直接环化形成,强化ATP合成有利于产物的积累。在分批发酵24h添加3g/L-broth丙酮酸钠(辅助能量物质),cAMP浓度达到4.13g/L,比对照批次提高了24.4%,发酵性能得到明显改善。对关键酶活性及能量代谢水平的测定结果表明,由于丙酮酸钠的添加,丙酮酸激酶的活性显著下降,而6-磷酸葡萄糖脱氢酶、琥珀腺苷酸合成酶和腺苷酸环化酶等产物合成途径中酶的活性均明显提高;异柠檬酸脱氢酶、琥珀酸脱氢酶和呼吸链脱氢酶等酶活性,以及辅因子NADH/NAD ⁺、ATP/AMP均明显提高。表明添加丙酮酸钠改变了糖酵解和磷酸戊糖途径间的碳流分配,使更多碳流向产物合成途径,同时提高了整体能量代谢水平,更利于ATP的生成,为产物的合成提供了物质和能量基础,进而促进了cAMP的合成与积累。     

辅助能量物质强化环磷酸腺苷发酵合成机制

微生物代谢过程中,环磷酸腺苷(cAMP)由ATP直接环化形成,强化ATP合成有利于产物的积累。在分批发酵24h添加3g/L-broth丙酮酸钠(辅助能量物质),cAMP浓度达到4. 13g/L,比对照批次提高了24. 4%,发酵性能得到明显改善。对关键酶活性及能量代谢水平的测定结果表明,由于丙酮酸钠的添加,丙酮酸激酶的活性显著下降,而6-磷酸葡萄糖脱氢酶、琥珀腺苷酸合成酶和腺苷酸环化酶等产物合成途径中酶的活性均明显提高;异柠檬酸脱氢酶、琥珀酸脱氢酶和呼吸链脱氢酶等酶活性,以及辅因子NADH/NAD+、ATP/AMP均明显提高。表明添加丙酮酸钠改变了糖酵解和磷酸戊糖途径间的碳流分配,使更多碳流向产物合成途径,同时提高了整体能量代谢水平,更利于ATP的生成,为产物的合成提供了物质和能量基础,进而促进了cAMP的合成与积累。     

低聚磷酸盐与次黄嘌呤偶合添加提高环磷酸腺苷发酵性能

环磷酸腺苷(cAMP)在微生物细胞内由ATP直接环化形成,而ATP的合成需要能量与前体的持续供应。通过添加次黄嘌呤激活补救途径,促进了cAMP的合成,与对照批次相比生产效率提高了39. 1%,但发酵进行至51h产物不再生成,而且产量未能得到提高。偶合添加次黄嘌呤和2g/L-broth六聚偏磷酸钠发酵批次的cAMP产量达到7. 24g/L,比单独添加次黄嘌呤和六聚偏磷酸钠的批次产量分别提高了125. 5%和93. 5%,生产效率也显著提高,达到了0. 101g/(L·h)。六聚偏磷酸钠和次黄嘌呤偶合添加工艺将低聚磷酸盐和补救途径的优势相结合,有效促进了cAMP合成与积累。     

长角血蜱唾液腺中腺苷三磷酸双磷酸酶的纯化及其酶切机制

利用染料亲和层析(Cibacorn Blue柱)和离子交换层析(Macrosphere WCX柱)对长角血蜱Haemaphysalis longicornis唾液腺的腺苷三磷酸双磷酸酶进行纯化,经SDS-PAGE证实其分子量为66 kD。腺苷三磷酸双磷酸酶可以水解ATP和ADP,但对AMP无水解作用,水解ATP和ADP的K_m值均为0.2 μmol/L,V_max值分别为12.5和15.6 μmol/(min·mg)。腺苷三磷酸双磷酸酶水解ATP的中间产物是ADP,最终产物是AMP和正磷酸。表明腺苷三磷酸双磷酸酶水解ATP的位点是5'-核苷酸的γ-磷酸键,水解ADP的位点是5'-核苷酸的β-磷酸键。     

硫胺素对Arthrobacter sp.A302环磷酸腺苷发酵的影响

研究在培养基中加入硫胺素（V B1）对cAMP发酵的影响。结果表明：V B1的最适添加量为0.5 g/L,与对照组相比,环磷酸腺苷（cAMP）产量和细胞干质量分别提高了36.4%和41.8%,达到7.5和7.8 g/L。琥珀酸和α-酮戊二酸的含量有明显的提高,平均提高了43.59%和40.77%;同时,主要副产物乙酸的含量没有明显变化。在发酵过程中,V B1的加入对ATP的含量及与cAMP合成密切相关的几种酶的活性也有显著的影响。     

环腺苷酸及其相关化合物的氧化铝薄板层析

使用极性不同的溶剂系,研究了3',5'-环腺苷酸(cAMP)及其相关化合物在国产中性氧化铝薄板上的层析行为,发现正丁醇:乙醇:水(30:10:13)系统能把cAMP与腺嘌呤、腺苷、3',5'-环鸟苷酸、二腺苷焦磷酸、AMP(GMP GTP ATP)等分离开来。磷酸盐和柠檬酸盐的中性水溶液能定量地洗脱cAMP斑点。从而使方法达到分离定量的目的,用于cAMP化学合成反应液的分析和环核苷酸磷酸二酯酶活力的测定。     

紫云英根瘤菌109菌株吸氢的诱导表达——核苷、核苷酸与烟酸的促进效应

核苷酸和烟酸的添加,使紫云英根瘤菌109氢酶吸氢活性表达增加。cAMP(1 mmol/L),烟酸(70 mmol/L)的存在,缓解了葡萄糖酸钠或果糖引起的吸氢活性阻遏,cAMP的解阻遏效应在年轻的菌体(48 h)表现较为明显。但以MB为受体的破碎细胞吸氢活性则未见增加,烟酸的促进效应受到氯霉素(40μg/ml)的抑制。其他核苷或核苷酸,如腺嘌呤,尿嘧啶,ATP,ADP,AMP,UMP,UTP都能促进吸氢活性的表达。诱导氢酶前,细胞ATP库已处于低水平,并保持稳定,添加琥珀酸盐后,ATP库水平提高,吸氢活性表达受抑。     

正十六烷对节杆菌发酵产环磷酸腺苷的影响

溶氧在节杆菌发酵产环磷酸腺苷(c AMP)过程中起着重要作用。本研究中,笔者首先对4种氧载体(正癸烷、正十二烷、正十四烷和正十六烷)的最佳添加浓度和添加时间进行筛选。结果表明:在0 h添加2%(体积分数)正十六烷促进c AMP生产效果最佳。在5 L发酵罐中添加2%(体积分数)正十六烷后,细胞干质量(DCW)和c AMP产量分别达到10. 85 g/L和8. 87 g/L,比对照分别提高了19. 4%和23. 2%。氧载体的加入提高了发酵液中的氧传递系数(K_La),降低了胞内NADH/NAD~+比率以及胞内三磷酸腺苷(ATP)水平,而对关键酶的酶活影响不显著。     

氧载体对小白链霉菌发酵生产ε-聚赖氨酸的影响

为了有效改善发酵体系中的溶氧水平,提高小白链霉菌Streptomyces albulus PD-1发酵生产ε-聚赖氨酸的能力,文中通过对氧载体的种类、最佳添加浓度以及添加时间进行筛选,最终确定在0 h添加0.5%(V/V)的正十二烷促进ε-聚赖氨酸生产效果最佳。在5 L发酵罐0 h添加0.5%的正十二烷进行批次补料发酵,ε-聚赖氨酸的产量和菌体干重分别可以达到(30.8±0.46)g/L和(33.8±0.29)g/L,较之对照组分别提高了31.6%和20.7%。ε-聚赖氨酸的产量和菌体干重的提高归因于0.5%正十二烷的添加促进发酵液中溶氧水平从23.8%提高到32%,同时发酵液中的一种主要副产物(聚二氨基丙酸)的含量下降31%。实验结果表明,正十二烷的添加可以提高S.albulus PD-1发酵液中的溶氧水平,抑制副产物的生成,促进ε-聚赖氨酸的合成。     

十六烷基三甲基溴化铵对枯草杆菌( B. subtilis )黄嘌呤缺陷型菌株产生腺苷的影响

在枯草杆菌中,嘌呤核柑酸合成途径中丰在AMP和GMP转变成IMP的环行途径,而在该菌的黄嘌呤缺陷型菌株中IMP至XMP的分枝途径被阻塞,同时在该菌株内又有强的5’一核苷酸酶活性,这使其可以生成腺苷（AR）,但反馈抑制是腺苷产率的一个重要影响因素,使用季铵盐阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）可改善细胞的渗透性,减少细胞内产物的反馈抑制,促进产物的分泌,结果表明,在发酵60h添加4％的CTAB能使产率从3．0g/l提高到3．6g/l,提高幅度达20％。     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

Regulation by zinc and adenosine 3',5'-cyclic monophosphate of growth and citric acid accumulation in Aspergillus niger.

The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they excrete citrate but do not proliferate. The phase alternative was controlled by the concentration of zinc: high zinc (2 muM) maintained growth phase, and a zinc 'deficiency' apparently signaled the ransition to the accumulating phase. Cyclic AMP affected the rates of growth and acidogenesis when added to cultures growing at low but not at high zinc: that is, cAMP did not induce the phase transition, zinc deficiency did, Cyclic AMP enhanced growth early in the fermentation, but at later stages the response of the mycelia to cAMP changed, and then cAMP inhibited growth. When citrate eventually began to accumulate cAMP augmented its sypthesis. The growth and acidogenic responses were quite specific to cAMP, and were sensitive to concentrations of about 1 muM. Cyclic AMP also either promoted or retarded the appearance of an unidentified yellow pigment. It is proposed that the growth and accumulating phases are distinct differentiated states, at least with respect to cAMP metabolism.     

Influence of aminophylline on the lipids in Microsporum gypseum.

The effect of aminophylline on the lipid synthesis of Microsporum gypseum has been examined. A decreased incorporation of [14C]acetate into lipids was observed when the cells were incubated for 1 h with aminophylline which was reflected in all the individual lipid fractions. However, cells grown with aminophylline in the growth medium exhibited increased levels of total phospholipids, which was probably due to a rise in intracellular cAMP as these cells exhibited 4-fold increased levels of cAMP. Decreased activity of phosphodiesterase by aminophylline accounts for the increased cAMP levels. Increased phospholipid content in aminophylline grown cells was further supported by the increased incorporation of [14C]acetate into phospholipids as well as increased activities of phospholipid biosynthetic enzymes in comparison to non-supplemented cells.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Production of cyclic AMP and adenosine by the brain and prothoracic glands of Manduca sexta

Relatively large amounts of cyclic AMP are produced by the prothoracic glands (source of the insect moulting hormone or moulting hormone percursor) of the tobacco hornworm, Manduca sexta. Pharate pupal glands produce more cyclic AMP than early fifth instar larval glands, and the addition of aminophylline enhances cyclic AMP accumulation. The much lower cyclic AMP level in the absence of aminophylline indicates the presence of potent cyclic AMP phosphodiesterase activity. Brains (sources of the prothoracicotropic hormone) also produce cyclic AMP but at a lower rate. Brains efficiently produce adenosine from ATP while β-ecdysone inhibits adenosine formation in early fifth instar larval brains. β-Ecdysone stimulates adenyl cyclase in brains of both stages when aminophylline and fluoride are present but has no effect on cyclic AMP accumulation in prothoracic glands. The absence of fluoride greatly reduces the amount of cyclic AMP produced by prothoracic glands when aminophylline is present. No cyclic AMP is accumulated in prothoracic glands when both fluoride and aminophylline are absent or in brains when fluoride is absent, notwithstanding the presence of aminophylline. Other insect tissues were also analysed for cyclic AMP production and none showed levels nearly as high as the prothoracic glands, suggesting a close relationship between cyclic AMP production and the function of the gland.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation.

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extracellular cyclic AMP and adenosine appearance in adipose tissue of Sus scrofa: effects of exercise

Cyclic AMP (cAMP) appears extracellularly in a variety of tissues including brain, liver, and kidney; whether it appears in adipose tissue and responds to physiological perturbation is unknown. The purpose of this study was to examine adipose tissue extracellular cAMP appearance and metabolism in situ and in vitro in physiologically challenged animals. Littermate swine were either sedentary or exercise trained on a treadmill for 3 months and subjected to acute exercise on experiment day. In situ, microdialysis probes in subcutaneous back fat were perfused before, during, and after animals performed 20 mins of acute exercise, and dialysate was analyzed for cAMP and adenosine. In vitro, isolated adipocytes were hormonally stimulated to provoke cAMP synthesis and efflux, and plasma membrane phosphodiesterase and 5'-nucleotidase activities were measured. Extracellular cAMP and adenosine levels in adipose tissue of sedentary swine averaged 5.2 +/- 1.7 and 863 +/- 278 nM, respectively. Exercise training tended to increase extracellular cAMP (11.3 +/- 1.7 nM) and reduce extracellular adenosine (438 +/- 303 nM), although neither change was statistically significant. Acute exercise caused a significant 3-fold and 16-fold increase in extracellular cAMP and adenosine, respectively, compared to rest. These changes occurred despite a 2- to 3-fold increase in adipose tissue blood flow during acute exercise. In vitro, cAMP efflux from exercise-trained swine was 42% greater than that from adipocytes of sedentary swine, yet adipocyte plasma membranes from exercise-trained and sedentary swine did not differ in maximal phosphodiesterase and 5'-nucleotidase activities. We conclude that cAMP appears extracellularly in swine adipose tissue and that the levels of extracellular cAMP and adenosine in intact swine adipose tissue are influenced by both acute and chronic exercise. The subsequent impact of the changes in these biochemicals on local cellular metabolism and growth remains to be determined.     

The cyclic AMP-dependent initiation of DNA synthesis by T51B rat liver epithelioid cells.

The brief rise in the cellular cyclic AMP content which occurs late in the prereplicative phases of rat hepatocytes in vivo and T51B rat liver epitheloid cells in vitro seems to be necessary for the initiation of DNA synthesis. Thus, the extracellular calcium-deprivation in T51B rat liver cells in culture which induces a late G-1 block is rapidly reversible (cells surge into S phase within one hour) either by creating a cyclic AMP surge by the addition of calcium or 3-isobutyl-1-methyl xanthine (a cyclic 3',5'-nucleotide phosphodiesterase inhibitor) or by the exogenous addition of low concentrations of cyclic AMP itself (i.e., 10(-8)-10(-5) M). On the other hand, prevention of the calcium-induced cyclic AMP surge by imidazole (a cyclic 3',5'-nucleotide phosphodiesterase activator) blocked the initiation of DNA synthesis by the calcium-deprived T51B cells.     

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.