Solubilization and reconstitution of the gastric H,K-ATPase.

Rabon, E.; Gunther, R D; Soumarmon, Annick; Bassilian, Sara; Lewin, M. J. M.; Sachs, George

doi:10.1016/s0021-9258(17)39232-3

Cited by 51 publications

(4 citation statements)

References 32 publications

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“…Several detergents can be used to solubilize H/K-ATPase (9, 15, 43, 44). In the present investigations, nOG proved to be useful for solubilization without significant inactivation of the H/K-ATPase activity, as has been previously reported (44). When membrane-bound H/K-ATPase was solubilized with 2% (w/v) nOG, the specific activity of the supernatant fraction was approximately 71% of that of the membrane-bound H/K-ATPase preparations, retaining the same turnover number (V/EP) of the membrane H/K-ATPase (Table 1).…”

Section: Electron Microscopic Observation Of Rotary-shadowed H/k-atpa...supporting

confidence: 82%

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

et al. 2003

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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.

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Section: Electron Microscopic Observation Of Rotary-shadowed H/k-atpa...supporting

confidence: 82%

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

et al. 2003

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“…Solubilization might be expected to remove smaller peptides and increase the specific activity. The specific activity of enzyme from the 7% Ficoll-34% sucrose (heavier) fraction did approximately double when solubilized with «-octyl glucoside and reconstituted into liposomes (Rabón et al, 1985). However, the heavier fraction is only about half as active as the lighter fraction to begin with, so the final specific activity of the reconstituted enzyme fell within the range cited for the five enzyme preparations used in this study.…”

supporting

confidence: 60%

Binding of the fluorescent substrate analog 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric hydrogen ion-potassium-ATPase: evidence for cofactor-induced conformational changes in the enzyme

Faller¹

1990

Biochemistry

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TNP-ATP binds to the gastric H,K-ATPase with a 4.6-fold increase in fluorescence intensity and 10-nm blue shift that indicate a relatively hydrophobic protein environment. The fluorescence enhancement saturates and is compatible with binding to a single class of specific nucleotide sites with Kd less than 25 nM and N = 3.4 +/- 0.9 nmol mg-1. Cofactors of the H,K-ATPase affect the fluorescence enhancement. K+ causes a rapid fluorescence quench by binding to a single class of sites with Kd = 3 mM. Mg2+ rapidly and completely reverses the K+ quench and then causes a slow fluorescence quench. The maximum enhancement is approximately halved by either Mg2+ or K+ in titrations with both protein and fluorophore. Therefore, TNP-ATP reports changes in protein environment compatible with cofactor-induced changes in the conformation of the enzyme.

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“…The much faster rate of the E2K to E,K transition in the , -ATPase than in the Na,K-ATPase may explain repeated failure to observe a K+-occluded form of the , -ATPase (Rabón et al, 1985;De Pont et al, 1985), and the rapid rate of passive Rb+-Rb+ exchange by the , -ATPase (Soumarmon et al, 1984) compared to the rate at which the Na,K-ATPase carries out Rb+-Rb+ exchange (Karlish & Stein, 1982). It is unnecessary for weak nucleotide binding to speed up the rate of the E2K to E,K transition in order for the E,E2 conformational change to participate in the mechanism of the , -ATPase.…”

Section: Discussionmentioning

confidence: 99%

Temperature dependence of the rates of conformational changes reported by fluorescein 5'-isothiocyanate modification of the hydrogen ion-potassium- and sodium-potassium-ATPases

Faller

Scheiner‐Bobis²,

Farley³

1991

Biochemistry

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Stopped-flow fluorometry has been used to measure the forward and reverse rates of the conformational change from E1 to E2 in the fluorescein-modified proton and sodium pumps (1) as a function of Na+ and K+ concentrations to verify the proposed mechanism of ion interaction with the enzymes and (2) as a function of temperature to gain insight into the nature of the conformational transition. (1) The fluorescence changes caused by Na+ and K+ are consistent with rapid competitive binding of the two ions to the E1 conformations of the enzymes followed by rate-limiting transitions between E1K and E2K. (2) Reaction coordinate diagrams for the E1K to E2K transitions in the H,K-ATPase and Na,K-ATPase are qualitatively similar. Enthalpy barriers to reaction are partially compensated by increased entropy in the transition states. However, there are striking quantitative differences between the two enzymes. The E2K to E1K reaction of the H,K-ATPase is more than 2 orders of magnitude faster (tau 1/2 = 6 ms at 22 degrees C) than the reverse rate of the Na,K-ATPase transition (tau 1/2 = 1.6 s), explaining repeated failure to detect a K(+)-"occluded" form of the H,K-enzyme. The E2K conformer of the Na,K-ATPase is 3 orders of magnitude more stable than E1K, while the E1K and E2K conformations of the H,K-ATPase are nearly equivalent energetically.

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Solubilization and reconstitution of the gastric H,K-ATPase.

Cited by 51 publications

References 32 publications

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

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

Binding of the fluorescent substrate analog 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric hydrogen ion-potassium-ATPase: evidence for cofactor-induced conformational changes in the enzyme

Temperature dependence of the rates of conformational changes reported by fluorescein 5'-isothiocyanate modification of the hydrogen ion-potassium- and sodium-potassium-ATPases

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