title: Topography of a vacuolar-type H+-translocating ATPase: chromaffin-granule membrane ATPase I.
abstract: 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.
authors.affiliation: Department of Biochemistry, University Medical School, Edinburgh, Scotland, U.K.
authors.forename: D K
authors.initials: DK
authors.lastname: Apps
authors.forename: J M
authors.initials: JM
authors.lastname: Percy
authors.forename: J R
authors.initials: JR
authors.lastname: Perez-Castineira
mesh_terms: D000818:Animals; D001345:Autoradiography; D001710:Biotin; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D001710:Biotin; D006180:Proton-Translocating ATPases