Reynafarje B, Brand M D, Lehninger A L
J Biol Chem. 1976 Dec 10;251(23):7442-51.
The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.
已采用一种新的实验方法评估了线粒体的H⁺/位点比率（即每对电子穿过呼吸链每个能量保守位点时排出的质子数）。在该方法中，向有氧、经鱼藤酮抑制且去能的线粒体中添加琥珀酸引发呼吸初期，同时测量氧气摄取速率和H⁺排出速率。在缬氨霉素存在的情况下，使用K⁺或Ca²⁺作为移动阳离子来消散膜电位，并使H⁺定量排出到培养基中。在未采取抑制磷酸盐摄取预防措施的情况下，用该方法观察到的H⁺/位点比率接近2.0，这与使用氧脉冲技术获得的值一致（米切尔，P.和莫伊尔，J.（1967年）《生物化学杂志》105卷，1147 - 1162页）。然而，当通过用N - 乙基马来酰亚胺抑制磷酸 - 氢氧根反向转运体或耗尽线粒体的内源性磷酸盐含量来消除磷酸盐移动时，始终观察到接近4.0的H⁺/位点比率。只要存在足够的K⁺（加缬氨霉素）或Ca²⁺，该比率就与琥珀酸浓度、线粒体蛋白浓度、6至8之间的pH值以及培养基的离子组成无关。内源性ATP水解或其他离子（腺嘌呤核苷酸、三羧酸、HCO₃⁻等）转运的特异性抑制剂未显示会影响观察到的H⁺/位点比率。此外，用α - 甘油磷酸替代琥珀酸，α - 甘油磷酸是在内膜外表面被氧化因而无需进入基质的底物，得到的H⁺/位点比率与琥珀酸相同。结论是，当消除磷酸盐移动时，线粒体电子传递的H⁺/位点比率可能接近4.0。
