title: Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase.
abstract: Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.
authors.affiliation: Biological Chemistry, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Abe
authors.forename: Shunji
authors.initials: S
authors.lastname: Kaya
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Imagawa
authors.forename: Mahito
authors.initials: M
authors.lastname: Kikumoto
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Oiwa
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Katoh
authors.forename: Michio
authors.initials: M
authors.lastname: Yazawa
authors.forename: Kazuya
authors.initials: K
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry; D004789:Enzyme Activation; D005753:Gastric Mucosa / Q000201:enzymology; D005960:Glucosides / Q000737:chemistry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D012995:Solubility; D012996:Solutions; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D003902:Detergents; D005960:Glucosides; D007527:Isoenzymes; D008565:Membrane Proteins; D012996:Solutions; C018619:octyl-beta-D-glucoside; D017506:H(+)-K(+)-Exchanging ATPase