Active Detergent-solubilized H+,K+-ATPase Is a Monomer

Dach, Ingrid; Olesen, Claus; Signor, Luca; Nissen, Poul; Maire, Marc le; Møller, Jesper; Ebel, Christine

doi:10.1074/jbc.m112.398768

Cited by 11 publications

(7 citation statements)

References 89 publications

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“…Differential centrifugation of gastric mucosa from pig stomachs, with slight modifications from procedures previously reported 24–26 , was used for vesicle isolation. To characterize the gastric H + /K + -ATPase in our sample, enzyme activity was detected by measuring the amount of inorganic phosphate released using the PiColorLock Gold Phosphate Detection System.…”

Section: Resultsmentioning

confidence: 99%

“…We performed the labeling experiment using Rabe-Tz at a pH of 2, 3, 4 and 7 and observed labeling of a protein with apparent molecular weight of 270kDa at pH=3 (Figure 3D). While a recent study proposes that the active form of the ATPase is the monomeric form (≈114kDa) 26 , extensive early research suggest the enzyme functions as a dimer or higher oligomer in conjunction with the β subunit 28–33 . Our finding suggests that active ATP4A is a dimer.…”

Section: Resultsmentioning

confidence: 99%

“…Gastric H+/K+-ATPase was isolated using a protocol adapted from Dach et al 2012 26 and Skrabanka et al 1984 25 . Fresh porcine stomachs were obtained from Innovative Research Inc (Novi, MI) and kept on ice during each step.…”

Section: Methodsmentioning

confidence: 99%

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Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting

Paresi

Liu

2016

Mol. BioSyst.

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Affinity probes are useful tools for determining molecular targets and elucidating mechanism of action for novel, bioactive compounds. In the case of covalent inhibitors, activity based probes are particularly valuable for ensuring acceptable selectivity margins. However, there is a variety of bioorthogonalchemisty reactions available for modifying compounds of interest with clickable tags. Here, we describe a direct comparison of tetrazine ligation and strain promoted azide-alkyne cycloaddition using benzimidazole based probes to bind their known target, the gastric proton pump, ATP4A. This study validates the use of chemical probes for target identification and illustrates the superior efficiency of tetrazine ligation for copper-free click systems. In addition, we have identified several novel binding partners of benzimidazole probes: Isoform 2 of deleted in malignant brain tumors 1 protein (DMBT1) and three uncharacterized proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting

Paresi

Liu

2016

Mol. BioSyst.

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“…μg/ml and 47 (± 5) μg/ml, respectively. Based on the molecular weight of an αβ complex of the H + ,K + -ATPase of 147,346 g mol -1 [50] and again assuming a single eosin molecule binds per protein complex, the half-saturating protein concentrations correspond to dissociation constants, Kd, of 0.075 (± 0.007) μM and 0.32 (± 0.03) μM in the E1 and E2 states, respectively.…”

Section: Eosin Binding To the Gastric H + K + -Atpase And Sarcoplasmmentioning

confidence: 99%

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Hossain¹,

Li²,

Zhang³

et al. 2020

Biochimica et Biophysica Acta (BBA) - Biomembranes

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A fluorescence ratiometric method utilising the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na + ,K +-, H + ,K +-and sarcoplasmic reticulum Ca 2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na + ,K +-and H + ,K +-ATPases compared to the Ca 2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na + ,K +-ATPase and the Ca 2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na + ,K +-ATPase than the E2 conformation, but in the case of the Ca 2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na + ,K +-and H + ,K +-ATPases, the E2-E1 transition of the Ca 2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na + ,K +-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na + , K +-and the H + ,K +-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

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“…Thus, representative members of the P-ATPase family, such as the sarcoplasmic reticulum Ca 2ϩ pump (8) and the Na ϩ /K ϩ pump (9), self-associate. The monomer of the sarcoplasmic reticulum Ca 2ϩ -ATPase and the ␣-␤ protomers of the Na ϩ /K ϩ -and H ϩ /K ϩ -ATPases are capable of performing all the steps of the reaction cycle (8,10,11). The human PM Ca 2ϩ -ATPase is a P2B-ATPase that resembles PM H ϩ -ATPases by having an extended C-terminal regulatory domain.…”

mentioning

confidence: 99%

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Justesen

Hansen

Martens

et al. 2013

Journal of Biological Chemistry

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Active Detergent-solubilized H+,K+-ATPase Is a Monomer

Cited by 11 publications

References 89 publications

Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting

Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

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Active Detergent-solubilized H+,K+-ATPase Is a Monomer

Cited by 11 publications

References 89 publications

Benzimidazole covalent probes and the gastric H+/K+-ATPase as a model system for protein labeling in a copper-free setting

Benzimidazole covalent probes and the gastric H+/K+-ATPase as a model system for protein labeling in a copper-free setting

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

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Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting

Benzimidazole covalent probes and the gastric H⁺/K⁺-ATPase as a model system for protein labeling in a copper-free setting