Cidon S, Nelson N
J Biol Chem. 1986 Jul 15;261(20):9222-7.
An N-ethylmaleimide-sensitive ATPase was purified 100-fold from chromaffin granule membranes. The purification procedure included solubilization with polyoxyethylene 9 lauryl ether, chromatography on hydroxylapatite and DEAE-cellulose columns, and glycerol gradient centrifugations. Inclusion of phosphatidylserine and a mixture of protease inhibitors during the purification procedure was necessary to maintain the activity of the preparation. The purified preparation contained four major polypeptides with molecular masses of about 115, 72, 57, and 39 kDa, which were copurified with the ATPase activity. The 115-kDa subunit binds [14C]dicyclohexylcarbodiimide and the subunits of 115 and 39 kDa bind [14C]N-ethylmaleimide. The ATP-dependent proton uptake activity of chromaffin granule membranes is inhibited 50% with about 20 microM N-ethylmaleimide, while over 5 mM concentrations of the inhibitor were required to block the ATPase activity of the membranes. The ATPase activity of the purified enzyme was inhibited via two different affinities: a high affinity site with a Ki in the microM range and a low affinity site in the mM range, each contributing to about 50% inhibition of the enzyme. It is concluded that the proton-ATPase of chromaffin granule membranes contains at least four subunits with the 115-kDa polypeptide being the main subunit having the active site for the ATPase activity of the enzyme.
从嗜铬粒细胞膜中纯化出一种对N - 乙基马来酰亚胺敏感的ATP酶，纯化倍数达100倍。纯化步骤包括用聚氧乙烯9十二烷基醚增溶、在羟基磷灰石和二乙氨基乙基纤维素柱上进行层析以及甘油梯度离心。在纯化过程中加入磷脂酰丝氨酸和蛋白酶抑制剂混合物对于维持制剂的活性是必要的。纯化后的制剂包含四种主要多肽，分子量分别约为115、72、57和39 kDa，它们与ATP酶活性一起被共纯化。115 kDa亚基结合[14C]二环己基碳二亚胺，115和39 kDa亚基结合[14C]N - 乙基马来酰亚胺。嗜铬粒细胞膜的ATP依赖性质子摄取活性在约20 μM N - 乙基马来酰亚胺作用下被抑制50%，而阻断膜的ATP酶活性则需要超过5 mM浓度的该抑制剂。纯化酶的ATP酶活性通过两种不同亲和力被抑制：一种高亲和力位点，其Ki在微摩尔范围内，一种低亲和力位点在毫摩尔范围内，每种亲和力对酶抑制作用的贡献约为50%。结论是嗜铬粒细胞膜的质子 - ATP酶至少包含四个亚基，其中115 kDa多肽是具有酶ATP酶活性活性位点的主要亚基。
