Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector
Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector
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DOI：
10.1529/biophysj.107.111781
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发表时间：
2008-01-15
影响因子：
3.4
通讯作者：
Marsh, Derek
中科院分区：
文献类型：
--
作者：
Dixon, Neil;Pali, Tibor;Marsh, Derek
破骨细胞变种的空泡H+-ATPase(V-ATPase)是一种潜在的治疗靶点，可用于对抗与骨质疏松有关的过度骨吸收。在一系列基于5-(5，6dichloro-2-indolyl)-2-methoxy-2，4-pentadienamide的合成抑制剂(INDOL0)中，最有效的是证明了对破骨细胞酶的特异性，而不是其他V-ATPase。本文利用褐肾鱼肝胰腺c亚单位具有转运活性的16 kDa蛋白脂类似物，结合电子顺磁共振(EPR)谱，研究了两种氮氧自旋标记的衍生物(INDOL6和INDOL5)与V-ATPase的相互作用。用酿酒酵母的液泡膜也进行了类似的实验，其中V-ATPase的c亚基在功能上被Nephrops 16-kDa蛋白脂取代。INDOL5衍生物旨在优化EPR检测与V-ATPase的相互作用。在Nephrops 16-kDa蛋白脂的膜制剂中，INDOL5的EPR谱包含一个运动受限组分，这是由于吲哚类抑制剂与蛋白脂亚单位c的跨膜域直接结合而产生的。在液泡膜中的INDOL6衍生物的EPR谱中检测到类似的、但相当小的运动受限组分，除了在膜的流体双层区检测到较大的INDOL6组分外。高效的经典V-ATPase抑制剂刀豆素A在高浓度时诱导INDOL5的运动限制，这掩盖了较低浓度的刀豆素A置换的光谱效应。与母体INDOL0抑制剂最接近的INDOL6衍生物对破骨细胞V-ATPase显示出有限的亚型特异性，IC50在10纳摩尔范围内。
The osteoclast variant of the vacuolar H+-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC50 in the 10-nanomolar range.