钌红敏感的钙离子转运在线粒体中的定位及其在大鼠肝脏特定线粒体群体中富集的证据。

Submitochondrial location of ruthenium red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria.

作者信息

Bygrave F L, Heaney T P, Ramachandran C

出版信息

Biochem J. 1978 Sep 15;174(3):1011-9. doi: 10.1042/bj1741011.

DOI:10.1042/bj1741011

PMID:728072

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1186007/

Abstract

Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca(2+) transport in the absence and in the presence of P(i) as well as in the presence of N-ethylmaleimide to prevent P(i) cycling, succinate-supported respiration in the absence and in the presence of ADP, the DeltaE and -59 DeltapH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, alpha-glycerophosphate dehydrogenase, P(i) content and the effect on the ;resting' rate of respiration of repeated additions of a fixed Ca(2+) concentration. 2. Ca(2+) transport either in the presence or in the absence of added P(i) and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for DeltaE and -59 DeltapH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca(2+) transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca(2+), as measured by the effect of repeated additions of Ca(2+) on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of alpha-glycerophosphate dehydrogenase was similar to that of Ca(2+) transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem.25, 379-396], it is concluded that the Ruthenium Red-sensitive Ca(2+)- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca(2+) transport and the biogenesis of mitochondria is discussed.

摘要

通过在缓冲等渗蔗糖溶液中进行差速离心，从大鼠肝脏匀浆中制备出沉降系数在3000至120000g·min之间的七个组分。对这些组分分别进行了以下测量：在有无无机磷酸（P(i)）以及存在N-乙基马来酰亚胺以防止P(i)循环的情况下，对钌红敏感的钙离子（Ca(2+)）转运；在有无二磷酸腺苷（ADP）的情况下，琥珀酸支持的呼吸作用；质子动力势的ΔE和-59ΔpH组分；细胞色素氧化酶；解偶联剂刺激的三磷酸腺苷酶；α-甘油磷酸脱氢酶；P(i)含量；以及重复添加固定浓度的Ca(2+)对“静息”呼吸速率的影响。2. 在添加或不添加P(i)以及存在N-乙基马来酰亚胺的情况下，Ca(2+)转运在沉降系数为8000g·min时的组分中表现出显著更高的速率。相比之下，在添加或不添加ADP的情况下的呼吸作用以及在沉降系数在4000至20000g·min之间的组分中的ΔE和-59ΔpH值相似，这表明这些组分中Ca(2+)转运的驱动力相似。3. 通过重复添加Ca(2+)对静息呼吸速率的影响来测定各组分对Ca(2+)的容纳能力的实验表明，组分2，即沉降系数为8000g·min的组分，对该离子的解偶联作用也表现出最大的耐受性。4. 在三种酶活性图谱中，只有α-甘油磷酸脱氢酶的图谱与Ca(2+)转运的图谱相似。由于先前的研究人员已将该酶定位在内周膜的位点[Werner & Neupert (1972) Eur. J. Biochem. 25, 379 - 396]，因此得出结论，对钌红敏感的Ca(2+)转运系统也位于内膜的这一区域。讨论了这些发现与线粒体Ca(2+)转运机制和线粒体生物发生的关系。