Concanamycin a, the specific inhibitor of V-ATPases, binds to the Vo subunit c
Concanamycin a, the specific inhibitor of V-ATPases, binds to the Vo subunit c
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DOI：
10.1074/jbc.m207345200
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发表时间：
2002-10-25
影响因子：
4.8
通讯作者：
Wieczorek, H
中科院分区：
文献类型：
--
作者：
Huss, M;Ingenhorst, G;Wieczorek, H
从烟草天蛾（Manduca sexta）中肠纯化得到的V-ATP酶（V-ATP酶）被10 nm的V-ATP酶特异性抑制剂Plecomacrolide concanamycin A抑制50%。为了确定酶复合物中抗生素的结合位点，用半合成的9-O-[p-（三氟乙基二氮杂环丙烯基）-苯甲酰基]-21，23-二脱氧-23-[I-125]碘-康帕霉素A（J-康帕霉素A）进行标记，其在15-20 μ M的浓度下仍然抑制V-ATP酶50%。在用UV光处理后，从该康卡那霉素衍生物产生高反应性卡宾，导致与酶形成共价键。此外，放射性示踪剂1251使得标记的亚基的检测可行。V-1/V-O全酶，V-O复合物，和V-ATP酶含有杯状细胞顶端膜与concanadine的治疗导致的质子转运V-O复合物的蛋白脂质，亚基C的标记。J-伴卡那霉素A与亚基c的结合被伴卡那霉素A以浓度依赖性方式阻止，表明标记是特异性的。巴弗洛霉素A（1）和B-1也分别阻止了结合，但苯内酯烯酰胺水杨卤酰胺（一种新型V-ATP酶抑制剂）不阻止结合。
Vacuolar-type ATPase (V-ATPase) purified from the midgut of the tobacco hornworm Manduca sexta is inhibited 50% by 10 nm of the plecomacrolide concanamycin A, the specific inhibitor of V-ATPases. To determine the binding site(s) of that antibiotic in the enzyme complex, labeling with the semisynthetic 9-O-[p-(trifluoroethyldiazirinyl)-benzoyl]-21,23-dideoxy-23-[I-125]iodo-concanolide A (J-concanolide A) was performed, which still inhibits the V-ATPase 50% at a concentration of 15-20 mum. Upon treatment with UV light, a highly reactive carbene is generated from this concanamycin derivative, resulting in the formation of a covalent bond to the enzyme. In addition, the radioactive tracer 1251 makes the detection of the labeled subunit(s) feasible. Treatment of the V-1/V-o holoenzyme, the V-o complex, and the V-ATPase containing goblet cell apical membranes with concanolide resulted in the labeling of only the proteolipid, subunit c, of the proton translocating V-o complex. Binding of J-concanolide A to subunit c was prevented in a concentration-dependent manner by concanamycin A, indicating that labeling was specific. Binding was also prevented by the plecomacrolides bafilomycin A(1) and B-1 respectively, but not by the benzolactone enamide salicylihalamide, a member of a novel class of V-ATPase inhibitors.