Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate

Soumarmon, Annick; Robert, J; Lewin, M. J. M.

doi:10.1016/0005-2736(86)90504-3

Cited by 21 publications

(10 citation statements)

References 9 publications

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“…This protocol cross-links hemichannel connexin monomers while in plasma membrane, preserving their in situ structural relationships; subsequent subunit exchange is unlikely to occur. This controls for possible subunit exchange between solubilized hemichannels, as has been noted for a few oligomeric membrane proteins in the presence of detergent (57)(58)(59). After solubilization, the cross-linked material was immunoaffinity-purified for Cx32-containing structures, as before.…”

Section: Co-purification Of Connexin-32 and Connexin-26 -Connexinmentioning

confidence: 99%

Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules

Bevans¹,

Kordel²,

Rhee³

et al. 1998

Journal of Biological Chemistry

299

255

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Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

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Section: Co-purification Of Connexin-32 and Connexin-26 -Connexinmentioning

confidence: 99%

Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules

Bevans¹,

Kordel²,

Rhee³

et al. 1998

Journal of Biological Chemistry

299

255

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“…2). This may be due to inactivation of the active Ca-+ transporting enzyme in the presence of high levels of detergent which causes dissociation of oligomeric proteins (14,17,25) and the removal of phospholipids required for the activity (15,19).…”

Section: Discussionmentioning

confidence: 99%

Solubilization and Reconstitution of Ca²⁺ Pump from Corn Leaf Plasma Membrane

Kasai

Muto²

1991

Plant Physiol.

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“…However, further purification has proved difficult, largely because of loss of enzyme activity during the detergent solubilization and storage. Several classes of detergent, cationic (dodecyltrimethylammonium bromide [17]), anionic (SDS and cholate [16,18,19]) and non-ionic ooctylglucoside, Tween, Emulgen, octa(ethylene Abbreviations used : C 12 E 8 , octa(ethylene glycol)dodecyl monoether ; DOM, n-dodecyl-β-D-maltoside ; DTT, dithiothreitol ; CDTA, trans-1,2diaminocyclohexane-N,N,Nh,Nh-tetra-acetic acid ; NBD, 7-nitrobenz-2-oxa-1,3-diazole-4-yl. 1 To whom correspondence should be addressed (e-mail lacapere!curie.fr).…”

Section: Introductionmentioning

confidence: 99%

“…Key words : affinity chromatography, detergent solubilization, P-type ATPase. glycol)dodecyl monoether (C "# E ) ), Triton X-100, Nonidet P-40 and n-dodecyl-β--maltoside (DOM) [6,11,[17][18][19][20][21][22]q have been tested. Several types of biochemical technique (centrifugation, chromatography, etc.)…”

Section: Introductionmentioning

confidence: 99%

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Efficient solubilization and purification of the gastric H+,K+-ATPase for functional and structural studies

2000

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When gastric H(+),K(+)-ATPase-containing microsomes are solubilized by detergents, a rapid loss of ATPase activity is generally observed. In this article, SDS/PAGE of octa(ethylene glycol)dodecyl monoether (C(12)E(8))- and n-dodecyl beta-d-maltoside-solubilized microsomes and their purifications by affinity chromatography on Reactive Red column reveal that inactivation is due to two main effects. (i) Solubilization activates an aspartic protease that cleaves down the alpha-subunit of the H(+),K(+)-ATPase. Addition of pepstatin A at slightly acidic pH and at low temperature prevents the proteolysis. (ii) A too-harsh delipidation inactivates the ATPase. When n-dodecyl-beta-d-maltoside is the detergent, the soluble H(+), K(+)-ATPase is highly active (2.5 micromol/mg per h at pH 6.0 and 5 degrees C) as long as ATP is added. When C(12)E(8) is used, the detergent induces an inactivation due to delipidation, since addition of lipids restores activity. The two subunits of the H(+), K(+)-ATPase are present in equimolar ratio in the n-dodecyl beta-d-maltoside-purified complex. Moreover, two main types of complex (330 and 660 kDa) were resolved in non-denaturing gels and should be the dimeric (alphabeta)(2) and tetrameric (alphabeta)(4) heterodimers respectively. In conclusion, purification of active, stable, soluble complexes of H(+),K(+)-ATPase with few lipids (a lipid/protein ratio of 0.25, w/w) has been achieved. This material should be useful for further structural studies.

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Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate

Cited by 21 publications

References 9 publications

Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules

Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules

Solubilization and Reconstitution of Ca²⁺ Pump from Corn Leaf Plasma Membrane

Efficient solubilization and purification of the gastric H+,K+-ATPase for functional and structural studies

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Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate

Cited by 21 publications

References 9 publications

Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules

Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules

Solubilization and Reconstitution of Ca2+ Pump from Corn Leaf Plasma Membrane

Efficient solubilization and purification of the gastric H+,K+-ATPase for functional and structural studies

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Solubilization and Reconstitution of Ca²⁺ Pump from Corn Leaf Plasma Membrane