Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.
Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.
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富含高尔基体的膜的质子转位 ATP 酶的纯化和重建。
DOI：
10.1073/pnas.85.24.9590
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发表时间：
1988
影响因子：
11.1
通讯作者：
Al-Awqati,Q
中科院分区：
文献类型：
--
作者：
Young,GP;Qiao,JZ;Al-Awqati,Q
通过细胞分离分离出富含高尔基标记物的肾皮质微粒体，并发现其含有一种被n-乙基马来酰亚胺（NEM）抑制的质子易位atp酶。该nem敏感的atp酶用正辛基葡萄糖苷溶解，在DEAE-Sephadex和QAE-Sephadex柱上用阴离子交换筛选层析纯化，最后用羟基磷灰石高效液相色谱柱纯化。纯化后的酶比活性为4.4 μ mol.mg-1。min-1被NEM完全抑制。添加聚乳素和透析去除洗涤剂导致nem敏感电致质子转运的重建。该空泡atp酶由5个多肽组成，表观分子质量分别为68、58、40、37和16 kDa。
Kidney cortex microsomes enriched in Golgi markers and probably also containing endosomes were isolated by cell fractionation and found to contain a proton-translocating ATPase that was inhibited by N-ethylmaleimide (NEM). This NEM-sensitive ATPase was solubilized with n-octyl glucoside and purified using anion-exchange sievorptive chromatography on sequential DEAE-Sephadex and QAE-Sephadex columns followed by a final hydroxyapatite HPLC column. The purified enzyme, with a specific activity of 4.4 mumol.mg-1.min-1 was completely inhibited by NEM. Addition of asolectin and removal of the detergent by dialysis resulted in reconstitution of NEM-sensitive electrogenic proton transport. This vacuolar ATPase is composed of five polypeptides with apparent molecular masses of 68, 58, 40, 37, and 16 kDa.