Isolation of (αβ)4‐Tetraprotomer Having Half‐of‐Sites ATP Binding from Solubilized Dog Kidney Na+/K+‐ATPase

Hayashi, Yutaro; Shinji, Nobuko; Tahara, Yoshikazu; Hagiwara, Emi; Takenaka, Hiroyuki

doi:10.1111/j.1749-6632.2003.tb07167.x

Cited by 8 publications

(9 citation statements)

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“…k The value of 3.51 ( 0.13 units/mg was used for normalization. mol of protomer in T, D, and P, respectively, at 1 µM ATP (17). Thus, considering the stoichiometry for the ATPbinding site, it can be reasonably concluded that the specific activity ratio of T, D, and P is 1:2:2.…”

Section: Discussionmentioning

confidence: 95%

Isolation of Stable (αβ)₄-Tetraprotomer from Na⁺/K⁺-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

et al. 2008

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Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) >> Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.

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Section: Discussionmentioning

confidence: 95%

Isolation of Stable (αβ)₄-Tetraprotomer from Na⁺/K⁺-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

et al. 2008

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“…The solubilization of active H/K-ATPase using C 12 E 8 at 5 °C has been reported (15). However, further studies are required to obtain an active solubilized H/K-ATPase with C 12 E 8 at room temperature as in the case of other P-type ATPases (31,42).…”

Section: Discussionmentioning

confidence: 99%

“…To assess the distribution of oligomeric species of C 12 E 8 -solubilized H/K-ATPase, which showed very low H/K-ATPase activity and phosphorylation capacity (Table 1), oligomeric H/K-ATPases were separated by HPGC (Figure 2A). When the C 12 E 8solubilized H/K-ATPase was applied to the column, four major protein fractions appeared, an aggregate (A), higher oligomers (H), diprotomers (D), and a protomer (P), as observed in the case of Na/K-ATPase (40)(41)(42). Fraction A was too large to permit an estimation of molecular weight (void volume of the column).…”

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

confidence: 99%

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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“…The rate constant of EP formation was shown to be at least 20-fold larger than that of breakdown and the rate constant for EP formation is known to increase with increasing concentrations of ATP (19,21). The reduced apparent affinity for ATP seen in steady state phosphorylation measurements (Fig.…”

Section: Larger Apparent Affinity Decrease For Atp Effects Of Each Mumentioning

confidence: 99%

“…Each mutation in the conserved GDASE sequence induced different affinity changes in the two different ATP effects and the affinity for pNPP providing further support for the presence of a single ATP binding site/␣-chain as proposed (12,37,43). Quite recently, three interesting studies appeared, the association of ␤Ϫ␤ chain (51) and the association of expressed ␣-chains by MgATP (54) and the specific activity of the Na/K-ATPase activity of the tetraprotomeric Na/K-ATPase fraction was approximately half that of the diprotomeric and protomeric fractions, using a solubilized dog kidney enzyme (21), which nicely explains both that protomer is sufficient for Na/K-ATPase activity (46,48) and oligomericity is required for the enzyme (11-13, 19 -20, 37, 43, 51-52, 54). These data are consistent with a hypothesis in which each ␣-subunit contains 1 mol of a high affinity ATP binding site for EP formation and 1 mol of low affinity ATP binding site for EATP formation, in the 2n-mer, not only in Na/K-ATPase but also in H/K-ATPase, possibly as (EP:EATP) 2 (21, 37, 43, 52).…”

Section: Larger Apparent Affinity Decrease For Atp Effects Of Each Mumentioning

confidence: 99%

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

Imagawa

Kaya

Taniguchi

2003

Journal of Biological Chemistry

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.5 ) and the apparent K m for Mg 2؉ , Na ؉ , K ؉ , and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was ϳ2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20ϳ50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, K m l /K m h , for each mutant was 1.3ϳ3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each K m P value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442 GDASE 446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg 2؉ , Na ؉ , and K ؉ effects.

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Isolation of (αβ)₄‐Tetraprotomer Having Half‐of‐Sites ATP Binding from Solubilized Dog Kidney Na⁺/K⁺‐ATPase

Cited by 8 publications

References 1 publication

Isolation of Stable (αβ)₄-Tetraprotomer from Na⁺/K⁺-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

Isolation of Stable (αβ)₄-Tetraprotomer from Na⁺/K⁺-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

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

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

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Isolation of (αβ)4‐Tetraprotomer Having Half‐of‐Sites ATP Binding from Solubilized Dog Kidney Na+/K+‐ATPase

Cited by 8 publications

References 1 publication

Isolation of Stable (αβ)4-Tetraprotomer from Na+/K+-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

Isolation of Stable (αβ)4-Tetraprotomer from Na+/K+-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

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

The Amino Acid Sequence 442GDASE446 in Na/K-ATPase Is an Important Motif in Forming the High and Low Affinity ATP Binding Pockets

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Isolation of (αβ)₄‐Tetraprotomer Having Half‐of‐Sites ATP Binding from Solubilized Dog Kidney Na⁺/K⁺‐ATPase

Isolation of Stable (αβ)₄-Tetraprotomer from Na⁺/K⁺-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

Isolation of Stable (αβ)₄-Tetraprotomer from Na⁺/K⁺-ATPase Solubilized in the Presence of Short-Chain Fatty Acids