烧伤焦痂毒性物质D_1对大鼠肝线粒体H^+-ATP酶活性及能量偶联的影响

Effects of D 1 Extracted From Burn Eschar on H +-ATPase Activity and Energy Coupling of Liver Mitochondria in the Rats

目的观察烧伤焦痂Ｄ１成分对肝线粒体Ｈ＋－ＡＴＰ酶活性及ＡＴＰ生成的影响，以探讨Ｄ１对线粒体呼吸抑制的作用机制。方法制备大鼠肝亚线粒体成分并从烧伤焦痂中提取Ｄ１成分，在含线粒体的反应体系中加入Ｄ１成分，测Ｈ＋－ＡＴＰ酶活性及ＡＴＰ生成量，并检测ＡＮＳ荧光强度以反映线粒体氧化磷酸化偶联状态。结果亚线粒体颗粒ＡＴＰ酶水解活力的正常值为（７．１８±０．０６）μｍｏｌ·ｍｇ１·ｍｉｎ１，与Ｄ１作用后，Ｈ＋－ＡＴＰ酶活力有明显下降，其下降程度和Ｄ１的剂量呈负相关，并且本实验直接观察到在新鲜制备ＳＭＰ中Ｄ１成分，直接抑制了ＡＴＰ的生成。ＡＮＳ荧光强度下降，亦与Ｄ１剂量呈负相关。结论Ｈ＋－ＡＴＰ酶活力下降可能是Ｄ１对线粒体呼吸抑制机制之一，Ｈ＋－ＡＴＰ酶活力下降导致ＡＴＰ生成减少。荧光强度下降反映线粒体氧化磷酸化功能下降。

Objective Burn eschar contains many toxic substances. The present study was aimed to study the effects of D 1, a toxic component from burn eschar, on mitochndrial H +-ATPase activity and ATP production. Methods Submitochondria and D 1 were freshly prepared from rat liver and burn eschar separately; D 1 was added to the reaction medium containing submitochondria. The H +-ATPase activity, ATP content and ANS fluorescence intensity were measured. Results The control value of H-ATPase activity in submitochondria was (7.18±0.06) Piμmol/mg. H +-ATPase activity was reduced sharply after adding D 1 into reaction medium and there existed a negative correlation between D 1 concentration and H +-ATPase activity. Furthermore, it was directly observed in the present study that D 1 could inhibit ATP production in submitochondria. Decrease in ANS fluorescence intensity was negatively correlated with D 1 concentration. Conclusions Reduction of H +-ATPase activity may contribute to the inhibition of mitochondrial oxidative phosphorylation by D 1, and to the decrease of ATP production. Decline in ANS fluorescence intensity reflects the suppression in mitochondrial oxidative phosphorylation.