Adenine nucleotide translocation of mitochondria. Identification of carrier sites.
Adenine nucleotide translocation of mitochondria. Identification of carrier sites.
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线粒体的腺嘌呤核苷酸易位。
DOI：
10.1111/j.1432-1033.1970.tb01086.x
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发表时间：
1970
期刊：
影响因子：
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通讯作者：
M. Klingenberg
中科院分区：
文献类型：
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作者：
M. J. Weidemann;H. Erdelt;M. Klingenberg
本研究表明，在线粒体膜上存在ADP和ATP的特异性结合位点，这可以归因于腺嘌呤核苷酸载体。参数的结合和各种其他性质的载体网站的determined.1载体网站的分化是基于这样的假设，即腺苷从这些网站竞争性地删除腺嘌呤核苷酸。为了准确评估和区分载体，比较了三种平行结合样品：（a）总结合;（B）腺嘌呤核苷酸前的核苷加成，其防止与载体结合和与内源性腺嘌呤核苷酸的交换;（c）在腺嘌呤核苷酸之后添加腺苷酸，其从载体中置换腺嘌呤核苷酸，但不从交换的池中置换腺嘌呤核苷酸。（a）与载体结合（=特异性结合）;（B）交换;（c）与非特异性结合，对腺苷不敏感的位点。2已经开发了用于耗尽内源性腺嘌呤核苷酸的线粒体的程序，以增加与可交换的内源性腺嘌呤核苷酸库的大小相关的对腺苷可去除的结合的比例。与通过与内源性腺嘌呤核苷酸交换的“结合”相比，即使在0°下，可去除的位点也非常迅速。4通过用未标记的核苷酸竞争性取代[14 C]ADP测量的载体结合的特异性显示结合仅限于ATP、ADP、dADP，并且在较小程度上，腺苷四磷酸。高特异性与腺嘌呤核苷酸交换的测量结果一致。5根据特异性腺嘌呤核苷酸结合的浓度依赖性，获得了与两个亲和力不等的位点（Kd′= 0.3 μM;Kd”= 7 μM）一致的非线性Scatchard图。载体位点的总数为C 0/cyt.a= 1.3（摩尔/摩尔）。高、低亲和力型之比为C ′/C”= 1：5。大鼠心脏的相应值为：Kd′= 0.1 μM，Kd”= 4 μM; C 0 = 2.2;C′/C”= 1：4。与ATP的两个结合位点的亲和力不等也得到了。ATP的Kd ′和Kd“均比ADP高1.5 ~ 2倍。ATP结合不受解偶联剂的影响。6在砷耗竭的大鼠肝线粒体中发现一个高亲和力的结合位点（Kd= 0.5 μM），C 0/cyt.a= 1.2（mole/mole）。这只是在大鼠心脏中发现的含量（每细胞）的一半。7特异性和非特异性结合之间的一些区别也可能基于SH和组氨酸试剂。只有非特异性结合对氯汞苯甲酸盐敏感。特异性结合对亚甲蓝催化的光氧化选择性敏感。这一事实，以及pH 7.0和7.5之间可去除腺嘌呤核苷结合的强烈降低，表明组氨酸基团参与了特异性结合。8声波颗粒保留了一些摄取腺嘌呤核苷酸的能力，但这不再是对腺嘌呤核苷敏感的。总腺嘌呤核苷酸结合的浓度依赖性给出了非线性Scatchard图，其可以由具有不等亲和力的两个结合位点拟合。讨论了在完整线粒体中特定位点的声波颗粒中的β-“不敏感”位点的可能身份。用去污剂处理也在很大程度上去除了对侧敏感和对侧可去除的结合。9具有不同亲和力的两个位点的出现，这表明，反映了载体的内部和外部定位。高亲和力位点可能是那些与残留的内源性腺嘌呤核苷酸接触的位点，
The present study demonstrates the existence of specific binding sites for ADP and ATP at the mitochondrial membrane which can be attributed to the adenine nucleotide carrier. Parameters of the binding and various other properties of the carrier sites are determined.1The differentiation of the carrier sites is based on the assumption that atractyloside removes adenine nucleotides competitively from these sites. For the accurate evaluation and discrimination of the carriers three parallel binding samples are compared: (a) total binding; (b) atractyloside addition before adenine nucleotide, which prevents binding to the carrier and the exchange with endogenous adenine nucleotides; (c) addition of atractyloside after adenine nucleotide, which displaces adenine nucleotide from the carrier but not from the exchanged pool.Total uptake of adenine nucleotides is thus differentiated into three portions: (a) binding to the carrier sites (= specific binding); (b) exchange; (c) binding to unspecific, atractyloside‐insensitive sites.2Procedures have been developed for depleting mitochondria of endogenous adenine nucleotides to increase the proportion of the atractyloside‐removable binding in relation to the magnitude of the exchangeable endogenous adenine nucleotide pool.3Kinetics of the binding at atractyloside‐removable sites are very rapid compared to “binding” by exchange with endogenous adenine nucleotides, even at 0°.4The specificity of the carrier binding, as measured by competitive replacement of [14C]ADP with unlabelled nucleotides, shows binding confined to ATP, ADP, dADP and, to a lesser extent, adenosine tetraphosphate. The high specificity is in agreement with that measured for the adenine nucleotide exchange.5From the concentration dependence of the specific adenine nucleotide binding, non‐linear Scatchard plots are obtained consistent with two sites of unequal affinity (Kd′= 0.3 μM;Kd”= 7 μM). The total number of carrier sites isC0/cyt.a= 1.3 (mole/mole). The ratio of high/low affinity types isC′/C”= 1:5. The corresponding values for rat heart are:Kd′= 0.1 μM,Kd”= 4 μM;C0= 2.2;C′/C”= 1:4. With ATP two binding sites of unequal affinity are also obtained. BothKd′andKd”for ATP are 1.5 to 2 times higher than for ADP. ATP binding is not affected by uncouplers.6In arsenate depleted rat liver mitochondria a single binding site of high affinity (Kd= 0.5 μM) is found withC0/cyt.a= 1.2 (mole/mole). This is only half the content (per cyt.a) found in rat heart.7Some differentiation between specific and unspecific binding is also possible on the basis of SH and histidine reagents. Only the unspecific binding is sensitive top‐chloromercuribenzoate. The specific binding is selectively sensitive to methylene blue‐catalyzed photo‐oxidation. This fact, and the strong decrease in atractyloside‐removable binding between pH 7.0 and 7.5, suggests the involvement of a histidine group in the specific binding.8Sonic particles retain some ability for uptake of adenine nucleotides, but this is no longer atractyloside‐sensitive. The concentration dependence of total adenine nucleotide binding gives non‐linear Scatchard plots which can be fitted by two binding sites with unequal affinities. The possible identity of the atractyloside‐“insensitive” sites in sonic particles with the specific sites in intact mitochondria is discussed. Treatment with detergents also largely removes atractyloside‐sensitive and atractyloside‐removable binding.9The occurrence of two sites with different affinities, it is suggested, reflects inner and outer localizations of carriers. High affinity sites may be those which are in contact with residual endogenous adenine nucleotides and …