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 .

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

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 (r2=0.9971), the rate of glucose consumed (r2=0.9967) and the rate of pyruvate produced (r2=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 (r2=0.9958) and pyruvate kinase (r2=0.8706). These results are the first answer to the fundamental question of what controls the flux through glycolysis in Torulopsis glabrata.