氧化磷酸化抑制剂对光滑球拟酵母糖酵解速度的影响

Effect of Oxidative Phosphorylation Inhibitors on The Glycolytic Flux in Torulopsis glabrata

研究了不同浓度电子传递链抑制剂(鱼藤酮和抗霉素A) 和FOF1-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.

The relationship between the concentration of intracellular ATP and the glycolytic flux in Torulopsis glabrata was studied by adding oxidative phosphorylation inhibitors (rotenone, antimycin A and oligomycin). When 10 mg/L rotenone and antimycin A were added to the cell cultures, the concentrations of intracellular ATP were approximately 43% and 27.7% less than that of the control, respectively. The specific activity of phosphofructokinase, one of the rate limiting enzymes of the glycolytic pathway, increased by a factors of 3.4 and 2.3, in comparison of the control respectively. With the specific activity of phosphofructokinase increased, the rate of glucose consumed increased by a factor of 3.6 and 2.4 compared with the control, and the rate of pyruvate produced increased by 17% and 8.5% respectively. The specific activities of hexokinase and pyruvate kinase were not affected by the addition of rotenone or antimycin A. Furthermore, the concentration of intracellular ATP decreased by 64.3% upon addition of 0.05 mg/L oligomycin to the cell culture, and the growth of Torulopsis glabrata was ceased when 0.4 mg/L oligomycin was added to the culture broth at 24 h. Both the rate of glucose consumed and the rate of pyruvate produced were enhanced with increasing the concentration of oligomycin (<0.6 mg/L) in the cell cultures. As a result, the activity of phosphofructokinase (r(2)=0.9971), the rate of glucose consumed (r(2)=0.9967) and the rate of pyruvate produced (r(2)=0.965) were enhanced by a decrease in the energy level of the cell (the concentration of intracellular ATP). Increase of the rate of glucose consumed rooted in elevation of the specific activity of phosphofructokinase (r(2)=0.9958) and pyruvate kinase (r(2)=0.8706). These results are the first answer to the fundamental question of what controls the flux through glycolysis in Torulopsis glabrata.