Apps D K, Percy J M, Perez-Castineira J R
Department of Biochemistry, University Medical School, Edinburgh, Scotland, U.K.
Biochem J. 1989 Oct 1;263(1):81-8. doi: 10.1042/bj2630081.
Proteins exposed on the cytoplasmic face of isolated chromaffin granules were labelled by lactoperoxidase-catalysed radioiodination and by non-enzymic biotinylation. Granule membranes were then prepared, and the H+-translocating ATPase isolated by fractionation with Triton X-114. The labelling of individual ATPase subunits was assessed by polyacrylamide-gel electrophoresis, followed by autoradiography or by blotting and decoration with 125I-labelled streptavidin. Subunits of 72, 57 and kDa were strongly labelled, and could be removed from the membrane at pH 11: they are therefore extrinsic proteins. The 120 kDa subunit was also labelled, but it was not solubilized at pH 11. Photolabelling with a hydrophobic probe indicated that this subunit penetrates the bilayer, and enzymic degradation studies showed the presence of N-linked oligosaccharides; this subunit therefore spans the chromaffin-granule membrane. Labelling of the 17 kDa subunit occurred predominantly on the extracytoplasmic (matrix) face of the granule membrane. These results are consistent with this V-type ATPase having a structure that is generally similar to that of mitochondrial (F-type) ATPases, although the attachment of the 120 kDa subunit may be asymmetrical.
通过乳过氧化物酶催化的放射性碘化和非酶促生物素化对分离的嗜铬粒蛋白颗粒胞质面暴露的蛋白质进行标记。然后制备颗粒膜，并用Triton X-114分级分离法分离出H⁺转运ATP酶。通过聚丙烯酰胺凝胶电泳，随后进行放射自显影或印迹并用¹²⁵I标记的链霉亲和素进行显色，来评估各个ATP酶亚基的标记情况。72、57和kDa的亚基被强烈标记，并且在pH 11时可从膜上除去：因此它们是外在蛋白。120 kDa的亚基也被标记，但在pH 11时不溶解。用疏水探针进行光标记表明该亚基穿透双层，酶促降解研究显示存在N-连接寡糖；因此该亚基跨越嗜铬粒蛋白颗粒膜。17 kDa亚基的标记主要发生在颗粒膜的胞外（基质）面上。这些结果与这种V型ATP酶的结构总体上类似于线粒体（F型）ATP酶的结构一致，尽管120 kDa亚基的附着可能是不对称的。
