CHARACTERIZATION OF PHOSPHATE EFFLUX PATHWAYS IN RAT-LIVER MITOCHONDRIA
CHARACTERIZATION OF PHOSPHATE EFFLUX PATHWAYS IN RAT-LIVER MITOCHONDRIA
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DOI：
10.1042/bj2120279
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发表时间：
1983-01-01
影响因子：
4.1
通讯作者：
PEDERSEN, PL
中科院分区：
文献类型：
--
作者：
KAPLAN, RS;PEDERSEN, PL
在缬氨霉素和 KCl 存在的情况下添加 ATP 可以诱导完整线粒体的 H+-ATP 酶催化的 ATP 水解。这导致线粒体内 Pi 的增加，因此可以研究完整线粒体中潜在的 Pi 流出途径。将该方法与内部和外部 Pi 的直接测量相结合，尝试通过 Pi/H+ 和 Pi/二羧酸盐载体的经典操作和/或通过其他机制来确定 Pi 外流是否通过白术苷敏感转运蛋白发生。最初的实验重新审查了导致当前观点的证据，即 Pi 的一个外流途径是白术苷敏感的 ATP/ADP,0.5Pi 转运蛋白。没有发现证据支持这种外排途径。相反，在先前的研究中观察到的低Pi流出率（存在寡霉素）的白术苷敏感性是由于ATP进入众所周知的ATP/ADP转运系统，随后ATP水解和随后的Pi流出所致。因此，在这些条件下，ATP 水解并未完全受到抑制。 Pi 流出变得对白术苷敏感最有可能是因为该抑制剂阻止 ATP 进入，而不是因为它直接抑制 Pi 流出。在缬氨霉素和 K+（缺乏寡霉素）诱导的 ATP 水解产生高水平的基质 Pi 时，观察到 Pi 从大鼠肝线粒体中大量流出。这种流出的一部分可以被硫醇特异性试剂抑制，其浓度通常抑制 Pi/H+ 和 Pi/二羧酸盐载体。然而，即使在对氯汞苯甲酸酯、N-乙基马来酰亚胺加正丁基丙二酸酯或 mersalyl 存在的情况下，仍有很大一部分流出。对 mersalyl 不敏感的 Pi 流出，对羧基苍术苷也不敏感，是一个饱和过程，因此表明载体介导。在此流出过程中，线粒体内膜对其他低分子量阴离子（即苹果酸、2-酮戊二酸）保持相当大的不渗透性。这里提出的结果排除了白术苷敏感的 ATP/ADP,0.5Pi 转运系统作为大鼠肝线粒体中 Pi 流出的机制。相反，Pi 流出似乎发生在经典的 Pi/H+ 传输系统上以及通过对 mersalyl 不敏感的饱和过程。对抑制剂不敏感的Pi流出可能发生在部分Pi/H+载体分子上，其存在状态不同于通常催化Pi流入的状态。或者，可以存在单独的Pi流出载体。
ATP hydrolysis catalyzed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, an attempt was made to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited. Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). Results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalyzing Pi influx. Alternatively, a separate Pi efflux carrier may exist.