Correlation between the Activities and the Oligomeric Forms of Pig Gastric H/K-ATPase †

Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C12E8) 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 C12E8-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C12E8 reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C12E8-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 R-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C12E8-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. The transport of H + and K + coupled hydrolysis of ATP is performed by H/K-ATPase 1 (1-3). The enzyme has been shown to be composed of an R (catalytic)-chain and a ‚-chain, the latter of which plays a role in membrane insertion and traffic (R‚-protomer). The catalytic subunits of P-type ATPases, such as gastric H/K-ATPase, Na/K- ATPase, and sarcoplasmic Ca/H-ATPase, have homologous primary structures (4-6). High-resolution monomeric crystal structures of Ca/H-ATPase have recently been reported,