CLATHRIN-COATED VESICLES CONTAIN AN ATP-DEPENDENT PROTON PUMP
CLATHRIN-COATED VESICLES CONTAIN AN ATP-DEPENDENT PROTON PUMP
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DOI：
10.1073/pnas.80.5.1300
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
BRANTON, D
中科院分区：
文献类型：
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作者：
FORGAC, M;CANTLEY, L;BRANTON, D
从小牛脑分离的网格蛋白包被的囊泡含有atp依赖的质子泵。通过测量[14C]甲胺分布来监测质子运动。用[14C]甲胺平衡的包被囊泡中加入Mg2+和ATP会产生4- 5倍的浓度梯度，对应于。delta。pH值在0.6-0.7个单位之间的介质和被包被的囊泡内的酸性。质子离子载体羰基氰化物对三氟甲氧基苯基腙（FCCP）可消除atp依赖性[14C]甲胺的摄取，羧基试剂N，N''-双环己基碳二亚胺可部分抑制其摄取，但Na+，K+- atp酶抑制剂strophanthidin （100 .mu）不受影响。M)和钒酸盐（10 .亩）。M)和线粒体atp酶抑制剂寡霉素(10 .mu。G /ml)和奥洛维汀（1 . μ G /ml）。GTP支持[14C]甲胺的摄取，而非不可水解的类似物5′-腺苷酰亚胺二磷酸。K+和valinomycin对膜电位的耗散刺激了[14C]甲胺的摄取，而FCCP和valinomycin均刺激了strophanthidin抗性atp酶的活性。这些结果与在网格蛋白包被的囊泡中存在电致、atp依赖的质子泵是一致的。这种质子泵可能在受体介导的内吞作用中必不可少的酸化事件中起作用。
Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+ and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a .DELTA.pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles. ATP-dependent [14C]methylamine uptake was abolished by the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and partially inhibited by the carboxyl reagent N,N''-dicyclohexylcarbodiimide but was unaffected by the Na+,K+-ATPase inhibitors strophanthidin (100 .mu.M) and vanadate (10 .mu.M) and the mitochondrial ATPase inhibitors oligomycin (10 .mu.g/ml) and aurovertin (1 .mu.g/ml). GTP, but not the nonhydrolyzable analog 5''-adenylyl imidodiphosphate, could support [14C]methylamine uptake. Dissipation of the membrane potential with K+ and valinomycin resulted in stimulation of [14C]methylamine uptake, whereas both FCCP and valinomycin stimulated the strophanthidin-resistant ATPase activity. These results are consistent with the existence of an electrogenic, ATP-dependent proton pump in clathrin-coated vesicles. This proton pump may play a role in the acidification events that are essential in receptor-mediated endocytosis.