Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria
Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria
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DOI：
10.1152/ajpcell.00146.2003
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发表时间：
2004-03-01
影响因子：
5.5
通讯作者：
Willis, WT
中科院分区：
文献类型：
--
作者：
Messer, JI;Jackman, MR;Willis, WT
碳水化合物的缺乏会加速骨骼肌的疲劳，但由于丙酮酸既为主线氧化提供了乙酰辅酶A，又为柠檬酸循环(CAC)提供了无性碳，其机制仍不清楚。因此，从大鼠混合骨骼肌分离的线粒体中，丙酮酸和CAC动力学参数被独立地定量。当丙酮酸或苹果酸在另一种底物的饱和浓度下逐步加入时，用极谱方法测量线粒体的耗氧率(J(O))。这些底物滴定是在固定的线粒体外ATP自由能状态(DeltaG(P))的生理范围内进行的，该状态是用肌酸激酶能钳建立的，也是在饱和[ADP]时进行的。线粒体J(O)的表观K-m，K-苹果酸为21~32um，表观K-m，K-丙酮酸为12~26um，底物K-m值均随DeltaG(P)的降低而增大。两种底物的V-max也随着DeltaGP的下降而增加，反映了J(O)的热力学控制。据报道，活体骨骼肌[苹果酸]是本研究中测定的K-m，K-苹果酸的10倍。与之形成鲜明对比的是，测定的Km、丙酮酸接近于报告的与糖原耗竭相关的肌肉接近衰竭时的[丙酮酸]。当在线性热力学力流关系(DeltaG(P)-J(O))的背景下评估数据时，DeltaG(P)-J(O)斜率基本上对体内观察到的范围内的[苹果酸]变化不敏感，但随着生理范围内[丙酮酸]的下降而显著下降。线粒体呼吸对生理范围内的[丙酮酸]变化特别敏感。相比之下，生理性的[苹果酸]对体外测得的线粒体丙酮酸氧化的影响很小，如果有的话。
Carbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate (J(o)) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (DeltaG(P)), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent K-m,K-malate for mitochondrial J(o) ranged from 21 to 32 muM, and the apparent K-m,K-pyruvate ranged from 12 to 26 muM, with both substrate K-m values increasing as DeltaG(P) declined. V-max for both substrates also increased as DeltaGP fell, reflecting thermodynamic control of J(o). Reported in vivo skeletal muscle [malate] are > 10-fold greater than the K-m,K-malate determined in this study. In marked contrast, the Km, pyruvate determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow (DeltaG(P)-J(o)) relationship, the DeltaG(P)-J(o) slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.