Significant increase of glycolytic flux in Torulopsis glabrata by inhibition of oxidative phosphorylation

This study was aimed at increasing the glycolytic flux of the multivitamin-auxotrophic yeast Torulopsis glabrata by disturbing oxidative phosphorylation. We examined two different strategies to impede oxidative phosphorylation. The first strategy was disruption of the activity of the electron transfer chain (ETC), by either of two approaches. One was separately adding, at 10 mg L1, specific inhibitors of complex I (rotenone) or of the bc1 complex (antimycin A) to the culture broth of T. glabrata CCTCC M202019, which resulted in significantly decreased intracellular ATP levels (43% and 27.7%) and significantly increased rates of glucose consumption (qs) and pyruvate production (qp); another approach was breeding a respiratory-deficient mutant RD-16, in which cytochromes aa3 and b in the ETC were deleted after ethidium bromide mutagenesis, to reduce the ETC activity constitutively. The second strategy was inhibiting F0F1-ATP synthase with 0.05 mM oligomycin. Also, a neomycin-resistant mutant with 65% decreased F0F1-ATPase activity was studied. With the two strategies, the specific activity of phosphofructokinase (R2=0.9971), the average specific glucose consumption rate (R2=0.9967) and the average specific pyruvate production rate (R2=0.965) were closely correlated with the intracellular ATP level, all of them being increased at a lower intracellular ATP level.     

光滑球拟酵母新霉素抗性株加速葡萄糖代谢

为进一步提高光滑球拟酵母发酵生产丙酮酸的生产强度,在能量代谢分析的基础上提出了降低ATP合成酶活性、但不影响NADH氧化的育种策略。通过亚硝基胍诱变,获得一株新霉素抗性突变株N07,该菌株F1ATPase活性降低65%、丙酮酸产量高于48gL且单位细胞消耗葡萄糖能力提高38%。添加双环己基碳二亚胺(DCCD)、叠氮钠(NaN3)、新霉素显著降低出发株F1ATPase活性但不影响突变株F1ATPase活性。突变菌株胞内ATP含量下降23.7%导致生长速率和最终菌体浓度(为出发菌株的76%)均低于出发菌株,但葡萄糖消耗速度和丙酮酸生产速度分别提高34%和42.9%,发酵周期缩短12h。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶、丙酮酸激酶和磷酸甘油醛激酶的活性提高了63.7%、28.8%和14.4%,电子传递链关键酶活性提高10%。结果表明降低真核微生物F1ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢。     

一株耐受低pH光滑球拟酵母RT-6的生理特性

摘要：【目的】 研究一株耐受pH 1.91的光滑球拟酵母 (Torulopsis glabrata) RT-6的生理特性。【方法】在不同的pH条件下,对比分析原菌CCTCC M202019和突变株RT-6胞内pH、胞内ATP水平、H+-ATPase酶活、膜脂肪酸组成和聚磷酸盐含量等生理学特性的差异。【结果】与原菌比较,突变株RT-6：(1)生物量和丙酮酸产量分别提高了60.6 %和85.4 % (56 h);(2) 在胞外pH5.0、4.5、4.0时,胞内pH极显著高于原菌;(3)胞内ATP、H+-ATP     

Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production

AIMS: To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. METHODS AND RESULTS: Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680-685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103.5 or 57.4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g l(-1) at 62 h (yield on glucose of 0.651 g g(-1)) in a 7-l jar fermentor. CONCLUSIONS: The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. SIGNIFICANCE AND IMPACT OF THE STUDY: Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites.     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

氧化磷酸化抑制剂对光滑球拟酵母糖酵解速度的影响

研究了不同浓度电子传递链抑制剂 ( 鱼藤酮和抗霉素 A) 和 F_OF₁-ATPase 抑制剂 ( 寡霉素 ) 对光滑球拟酵母胞内 ATP 水平、葡萄糖消耗速度、糖酵解途径关键酶的影响 . 在培养液中添加 10 mg/L 鱼藤酮和抗霉素 A ,相对于对照组,胞内 ATP 分别下降了 43% 和 27.7% ,使糖酵解关键酶磷酸果糖激酶 (PFK) 的活性分别提高 340% 和 230% ,从而导致葡萄糖消耗速度增加 360% 和 240% ,丙酮酸生成速度提高了 17% 和 8.5%. 改变胞内 ATP 水平并不影响糖酵解途径其他关键酶 HK 、 PK 活性 . 微量的寡霉素 (0.05 mg/L) 可使胞内 ATP 含量下降 64.3% ,当培养液中寡霉素浓度达到 0.4 mg/L 时,细胞不能继续生长,葡萄糖消耗速度和丙酮酸的生成速度却随着寡霉素浓度 ( 小于 0.6 mg/L) 的增加而增加 . 表明氧化磷酸化途径中, ATPase 决定着 ATP 的生成 . 降低胞内 ATP 含量能显著提高 PFK 活性 (r²=0.9971) ,葡萄糖消耗速度 (r²= 0.9967) 以及丙酮酸生产速度 (r²= 0.965) ,葡萄糖消耗速度的增加是糖酵解途径中关键酶 PFK 活性 (r² = 0.9958) 和 PK 活性 (r²= 0.8706) 增加所导致的 . 这一结果有利于揭示真核微生物细胞中氧化磷酸化与中心代谢途径 ( 酵解 ) 的关系 .     

一种基于途径分析提高丙酮酸产量的育种策略

为进一步提高光滑球拟酵母发酵生产丙酮酸的水平 ,在途径分析的基础上提出了一种组成型降低丙酮酸脱酸酶、但增强乙酰辅酶A合成酶活性的育种策略。通过亚硝基胍诱变 ,获得 1株乙酸需求型突变株CCTCCM2 0 2 0 19,在外加乙酸的培养基中表现出高于出发株 2 1%的丙酮酸生产能力和良好的遗传稳定性。检测突变株CCTCCM2 0 2 0 19中丙酮酸代谢相关酶的活性发现 :(1)丙酮酸脱羧酶活性降低了 4 0 % ;(2 )外加乙酸与否的条件下 ,乙酰辅酶A合成酶的活性分别提高了 10 3 5 %和 5 7 4 % ;(3)添加乙酸和突变对丙酮酸羧化酶、丙酮酸脱氢酶系、乙醇脱氢酶和乙醛脱氢酶的活性没有显著影响。在含有乙酸的培养基中突变株细胞干重比出发株高 2 1 7% ,可能是因为乙酰辅酶A合成酶活性的提高 ,补充了因丙酮酸脱羧酶活性降低而引起的胞质乙酰辅酶A短缺。在 7L罐中含有 6g L乙酸钠的培养基中发酵 6 2h ,丙酮酸产量达到 6 8 7g L ,对葡萄糖的产率为 0 6 5 1g g。     

Redirection of the NADH oxidation pathway in Torulopsis glabrata leads to an enhanced pyruvate production

This study aimed at increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from adenosine triphosphate (ATP)-production pathway (oxidative phosphorylation pathway) to non-ATP production pathway (fermentative pathway). Two respiratory-deficient mutants, RD-17 and RD-18, were screened and selected after ethidium bromide (EtBr) mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, cytochrome aa ₃ and b in electron transfer chain (ETC) of RD-18 and cytochrome b in RD-17 were disrupted. As a consequence, the activities of key ETC enzymes of the mutant RD-18, including F₀F₁-ATP synthase, complex I, complex I + III, complex II + III, and complex IV, decreased by 22.2, 41.6, 53.1, 23.6, and 84.7%, respectively. With the deficiency of cytochromes in ETC, a large amount of excessive cytosolic NADH was accumulated, which hampered the further increase of the glycolytic flux. An exogenous electron acceptor, acetaldehyde, was added to the strain RD-18 culture to oxidize the excessive NADH. Compared with the parent strain, the concentration of pyruvate and the glucose consumption rate of strain RD-18 were increased by 26.5 and 17.6%, respectively, upon addition of 2.1 mM of acetaldehyde. The strategy for increasing the glycolytic flux in T. glabrata by redirecting the NADH oxidation pathway may provide an alternative approach to enhance the glycolytic flux in yeast.     

ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic form of this parasite, transmitted by tsetse flies, is considered to be dependent on oxidative phosphorylation for ATP production. Indeed, its respiration was 55% inhibited by oligomycin, which is the most specific inhibitor of the mitochondrial F0/F1-ATP synthase. However, a 10-fold excess of this compound did not significantly affect the intracellular ATP concentration and the doubling time of the parasite was only 1.5-fold increased, suggesting that oxidative phosphorylation is not essential for procyclic trypanosomes. To further investigate the sites of ATP production, we studied the role of two ATP producing enzymes, which are involved in the synthesis of pyruvate from phosphoenolpyruvate: the glycosomal pyruvate phosphate dikinase (PPDK) and the cytosolic pyruvate kinase (PYK). The parasite was not affected by PPDK gene knockout. In contrast, inhibition of PYK expression by RNA interference was lethal for these cells. In the absence of PYK activity, the intracellular ATP concentration was reduced by up to 2.3-fold, whereas the intracellular pyruvate concentration was not reduced. Furthermore, we show that this mutant cell line still excreted acetate from d-glucose metabolism, and both the wild type and mutant cell lines consumed pyruvate present in the growth medium with similar high rates, indicating that in the absence of PYK activity pyruvate is still present in the trypanosomes. We conclude that PYK is essential because of its ATP production, which implies that the cytosolic substrate level phosphorylation is essential for the growth of procyclic trypanosomes.     

Accelerating glycolytic flux of Torulopsis glabrata CCTCC M202019 at high oxidoreduction potential created using potassium ferricyanide

This study aimed to increase the glycolytic flux of the multivitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from oxidative phosphorylation to membrane-bound ferric reductase. We added potassium ferricyanide as electron acceptor to T. glabrata culture broth at 20% dissolved oxygen (DO) concentration, which resulted in: (1) decreases in the NADH content, NADH/NAD(+) ratio, and ATP level of 45.3%, 60.3%, and 15.2%, respectively; (2) high activities of the key glycolytic enzymes hexokinase, phosphofructokinase, and pyruvate kinase, as well as high expression levels of the genes encoding these enzymes; and (3) increases in the specific glucose consumption rate and pyruvate yield of T. glabrata was by 45.5% and 23.1%, respectively. Our results showed that membrane-bound ferric reductase offers an alternative and efficient NADH oxidation pathway at lower DO concentration, which increases the glycolytic flux of T. glabrata.     

ATP generation during reduced inorganic sulfur compound oxidation by<Emphasis Type="Italic"> Acidithiobacillus caldus</Emphasis> is exclusively due to electron transport phosphorylation

The synthesis of adenosine 5-triphosphate (ATP) (increase in phosphorylation potential) during the oxidation of reduced inorganic sulfur compounds was studied in the moderately thermophilic acidophileAcidithiobacillus caldus (strain KU) (formerly Thiohacillus caldus). The phosphorylation potential increased during the oxidation of all reduced inorganic sulfur compounds tested compared with resting cells. The generation of ATP in whole cells was inhibited by the F0F1 ATPase inhibitor oligomycin, electron transport chain inhibitors, valinomycin and potassium ions. There was no increase in the phosphorylation potential, nor synthesis of ATP. in the absence of electron transport. An apparent lack of substrate-level phosphorylation was indicated by the lack of adenosine 5-phosphosulfate reductase in tetrathionate-grown At. caldus. Studies were also performed on the synthesis of ATP by membrane vesicles of At. caldus when presented with an artificial proton gradient. Complete inhibition of ATP synthesis in these vesicles occurred when they were loaded with N,N-dicyclohexylcarbodiimide (DCCD), but not when they were loaded with oligomycin, vanadate or electron transport chain inhibitors. The data presented here suggest that during the oxidation of reduced inorganic sulfur compounds by At. caldus, all ATP is synthesized by oxidative phosphorylation via a membrane-bound F0F1 ATPase driven by a proton gradient.     

Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD+ availability

This study aimed at increasing the pyruvate productivity from a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of NAD+. We examined two strategies for increasing availability of NAD+. To supplement nicotinic acid (NA), the precursor of NAD+; and to increase the activity of alcohol dehydrogenase integrating with addition acetaldehyde as exterior electron acceptor. The addition of 8 mg l(-1) NA to the fermentation medium resulted in a significant increase in the glucose consumption rate (48.4%) and the pyruvate concentration (29%). An ethanol-utilizing mutant WSH-13 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the alcohol dehydrogenase activity of the mutant WSH-13 increased about 110% and the mutant could utilize ethanol as the sole carbon source for growth (1.8 g l(-1) dry cell weight). When growing with glucose, the addition of 4 mg l(-1) acetaldehyde to the mutant WSH-13 culture broth led to a significant increase in the glucose consumption rate (26.3%) and pyruvate production (22.5%), but the ratio of NADH/NAD+ decreased to 0.22. Acetaldehyde did not affect the glucose and energy metabolism at high dissolved oxygen (DO) concentration. However, at lower DO concentration (20%), maintaining the acetaldehyde concentration in the mutant culture broth at 4 mg l(-1) caused an increased NAD+ concentration but a decreased NADH concentration. As a consequence, the pyruvate production rate, the pyruvate yield on glucose and the pyruvate concentration were 68, 44 and 45% higher, respectively, than the corresponding values of the control (without acetaldehyde). The strategy for increasing the glycolytic flux and the pyruvate productivity in T. glabrata by increasing the availability of NAD+ may provide an alternative approach to enhance the metabolites productivity in yeast.     

Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants.

We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.     

环境条件对丙酮酸分批发酵的影响

考察了搅拌转速、pH和温度对丙酮酸分批发酵的影响。高转速（500r／min）下,丙酮酸产率较高（71％）,但葡萄糖消耗速度较慢（1．23g／（L·h））;低转速（300r／min）下,细胞消耗葡萄糖的速度加快（1．95g/（L·h））,而丙酮酸产率（0．48％）却明显下降。将搅拌转速恒定在400r／min可在一定程度上获得较高的丙酮酸产率（0．62％）和葡萄糖消耗速度（1．66g／（L·h））。CaCO3调节pH时,较多碳流从丙酮酸节点转向α-酮戊二酸节点和细胞生长,最终丙酮酸产量比NaOH调节pH时的发酵结果低38．7％;NH3·H2O调节pH时最终细胞浓度和丙酮酸产量仅为NaOH调节时的77．8％和90．9％。pH5．5时最利于丙酮酸的合成。较高的发酵温度加速T.glabrata积累丙酮酸,但同时会导致α-酮戊二酸的提前积累;而较低的温度下甘油和α-酮戊二酸积累较少,丙酮酸发酵的最适温度为28～30℃。     

降低光滑球拟酵母电子传递链活性加速丙酮酸合成

光滑球拟酵母CCTCCM2 0 2 0 19经溴化乙锭诱变 ,挑选假阳性呼吸缺陷型菌株共 4 0株。对其中 7株丙酮酸产量提高的突变株进行发酵性底物 (葡萄糖 )和非发酵性底物 (甘油、乙酸 )的利用能力测试 ,鉴定得到 3株呼吸缺陷型突变株RD 16、RD 17和RD 18。相对于出发菌株 ,呼吸缺陷型突变株生长速率下降 ,最终菌体浓度降低 2 1%～2 9% ,胞内ATP含量下降 15 %～ 2 1% ,但单位细胞耗葡萄糖能力和单位细胞产丙酮酸能力分别提高了 2 0 7%～30 7%和 30 7%～ 5 5 5 %。进一步研究发现 ,呼吸缺陷型突变株线粒体复合体Ⅰ、Ⅰ Ⅲ、Ⅱ Ⅲ和Ⅳ的活性分别下降了 34%～ 4 1%、38 6 %～ 5 2 6 %、2 1%～ 2 5 %、15 0 %～ 6 30 % ,表明线粒体电子传递链氧化NADH的功能受到抑制。为使酵解产生的NADH正常氧化 ,在RD 18菌株的对数生长期流加 2 1mmol L外源电子受体乙醛。发现细胞合成丙酮酸能力提高 2 1 6 % ,且葡萄糖消耗速度明显加快 ,发酵周期缩短 14h。结果表明适当削弱能量代谢能够提高真核微生物中心代谢途径的速度     

异源表达NADH选择性氧化酶提高光滑球拟酵母酵解速率及丙酮酸生产强度

摘要：【目的】为进一步提高光滑球拟酵母(Torulopsis glabrata)葡萄糖代谢速率及丙酮酸生产强度。【方法】将源于荚膜胞浆菌(Histoplasma capsulatum)的编码选择性氧化酶的AOX1基因过量表达于T. glabrata中,获得了一株线粒体内NADH氧化途径发生改变且胞内总NADH 氧化酶活性提高1.8倍的重组菌株AOX。【结果】与出发菌株CON比较,细胞浓度以及发酵周期降低了20.3%和10.7%,而平均比葡萄糖消耗速率和丙酮酸合成速率分别提高了34.7%和54.1%。其原因     

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver Inhibition of oxidative phosphorylation as the primary action

1. In slices of rat liver, oligomycin inhibited the net transport of Na⁺ and K⁺ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions.2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin.3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value.4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycin-resistant ATP synthesis.5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.     

Effect of uncouplers and inhibitors of oxidative phosphorylation on the reduced and oxidized forms of mitochondiral ATPase.

A series of uncouplers and inhibitors of oxidative phosphorylation have been studied with regard to their effect on the hydrolytic activity of the reduced and oxidized forms of isolated or membrane-bound mitochondrial ATPase. Uncouplers (2,4-dinitrophenol, dicoumarol), which are also activators of the hydrolytic activity of ATPase, were more potent activators on the oxidized form of the enzyme. Inhibitors of oxidative phosphorylation (oligomycin, azide and amytal) had a more potent inhibitory effect on the hydrolytic activity of ATPase in its reduced form. Purified F1-ATPase, oligomycin insensitive in the oxidized form of the enzyme, became sensitive to oligomycin in the reduced form. An interpretation of the results suggests the presence of a mechanism that unifies the action of these different compounds on the synthesis and hydrolysis of ATP catalyzed by mitochondrial ATPase.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and l-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of l-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP]². When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of l-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in l-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.