Kunz W S, Kudin A, Vielhaber S, Elger C E, Attardi G, Villani G
Department of Epileptology, University Bonn Medical Center, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.
J Biol Chem. 2000 Sep 8;275(36):27741-5. doi: 10.1074/jbc.M004833200.
In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.
在本研究中，通过用特异性抑制剂氰化钾滴定细胞色素c氧化酶（COX），已在人皂素通透的肌纤维中测定了COX的通量控制系数和代谢储备能力。在存在底物谷氨酸和苹果酸的情况下，在ADP刺激的人骨骼肌纤维中观察到COX有2.3±0.2倍的过量能力。发现该值取决于线粒体底物供应。在谷氨酸、苹果酸和琥珀酸共同存在的情况下，支持了约1.4倍更高的呼吸速率，此时仅测定出COX有1.4±0.2倍的过量能力。与这些发现一致，在三种底物存在的情况下，COX的通量控制从0.27±0.03增加到0.36±0.08。这些结果表明在人体骨骼肌中COX对呼吸有严格的体内控制。这种严格控制可能对线粒体肌病有重要影响。为支持这一结论，对两名患有慢性进行性外眼肌麻痹的患者的骨骼肌纤维进行分析，他们的线粒体DNA分别有11%和49%的缺失，结果显示COX的储备能力大幅降低，通量控制系数增加，表明该酶对氧化磷酸化有严重的速率限制。
