Moriyama Y, Nelson N
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Biochim Biophys Acta. 1989 Apr 14;980(2):241-7. doi: 10.1016/0005-2736(89)90405-7.
Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.
利用冷失活、与来自嗜铬颗粒的H⁺ -ATP酶各亚基抗体的免疫交叉反应以及用N,N'-二环己基[¹⁴C]碳二亚胺进行化学修饰等方法，对溶酶体H⁺ -ATP酶的亚基结构进行了研究。当在0℃下于氯化物或硝酸盐以及MgATP存在的条件下孵育时，溶酶体H⁺ -ATP酶会被不可逆地抑制。冷失活导致几种多肽（72、57、41、34和33 kDa）从膜上释放出来，这些多肽与嗜铬颗粒H⁺ -ATP酶的相应亚基具有相同的电泳迁移率。抗体的交叉反应表明，72、57和34 kDa的多肽在免疫学上与嗜铬颗粒H⁺ -ATP酶的相应亚基相同。抑制液泡ATP酶中质子转运的二环己基碳二亚胺主要标记了两种18和15 kDa的多肽，它们构成了该酶的膜部分。这些结果表明，溶酶体H⁺ -ATP酶是一种多聚体酶，其亚基结构与嗜铬颗粒H⁺ -ATP酶相似。还展示了通过冷失活和免疫交叉反应揭示的其他液泡H⁺ -ATP酶的亚基结构。
