Scholte H R, Yu Y, Ross J D, Oosterkamp I I, Boonman A M, Busch H F
Department of Biochemistry, Erasmus University, Rotterdam, The Netherlands.
Mol Cell Biochem. 1997 Sep;174(1-2):61-6.
We modified the isolation procedure of muscle and heart mitochondria. In human muscle, this resulted in a 3.4 fold higher yield of better coupled mitochondria in half the isolation time. In a preparation from rat muscle we studied factors that affected the stability of oxidative phosphorylation (oxphos) and found that it decreased by shaking the preparation on a Vortex machine, by exposure to light and by an increase in storage temperature. The decay was found to be different for each substrate tested. The oxidation of ascorbate was most stable and less sensitive to the treatments. When mitochondria were stored in the dark and the cold, the decrease in oxidative phosphorylation followed first order kinetics. In individual preparations of muscle and heart mitochondria, protection of oxidative phosphorylation was found by adding candidate stabilizers, such as desferrioxamine, lazaroids, taurine, carnitine, phosphocreatine, N-acetylcysteine. Trolox-C and ruthenium red, implying a role for reactive oxygen species and calcium-ions in the in vitro damage at low temperature to oxidative phosphorylation. In heart mitochondria oxphos with pyruvate and palmitoylcarnitine was most labile followed by glutamate, succinate and ascorbate. We studied the effect of taurine, hypotaurine, carnitine, and desferrioxamine on the decay of oxphos with these substrates. 1 mM taurine (n = 6) caused a significant protection of oxphos with pyruvate, glutamate and palmitoylcarnitine, but not with the other substrates. 5 mM L-carnitine (n = 6), 1 mM hypotaurine (n = 3) and 0.1 mM desferrioxamine (n = 3) did not protect oxphos with any of the substrates at a significant level. These experiments were undertaken in the hope that the in vitro stabilizers can be used in future treatment of patients with defects in oxidative phosphorylation.
我们改进了肌肉和心脏线粒体的分离程序。在人体肌肉中，这使得在一半的分离时间内，更好偶联的线粒体产量提高了3.4倍。在大鼠肌肉的制备物中，我们研究了影响氧化磷酸化（oxphos）稳定性的因素，发现通过在涡旋仪上振荡制备物、暴露于光以及储存温度升高，氧化磷酸化会降低。发现每种测试底物的衰减情况不同。抗坏血酸的氧化最稳定，对这些处理不太敏感。当线粒体在黑暗和低温下储存时，氧化磷酸化的降低遵循一级动力学。在肌肉和心脏线粒体的个体制备物中，通过添加候选稳定剂，如去铁胺、拉扎罗ids、牛磺酸、肉碱、磷酸肌酸、N-乙酰半胱氨酸、Trolox-C和钌红，发现氧化磷酸化得到了保护，这意味着活性氧和钙离子在低温下对体外氧化磷酸化损伤中起作用。在心脏线粒体中，丙酮酸和棕榈酰肉碱的氧化磷酸化最不稳定，其次是谷氨酸、琥珀酸和抗坏血酸。我们研究了牛磺酸、次牛磺酸、肉碱和去铁胺对这些底物氧化磷酸化衰减的影响。1 mM牛磺酸（n = 6）对丙酮酸、谷氨酸和棕榈酰肉碱的氧化磷酸化有显著保护作用，但对其他底物没有。5 mM L-肉碱（n = 6）、1 mM次牛磺酸（n = 3）和0.1 mM去铁胺（n = 3）对任何底物的氧化磷酸化都没有显著保护作用。进行这些实验是希望体外稳定剂将来可用于治疗氧化磷酸化缺陷的患者。
