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Dach I.,Center for Membrane Pumps in Cells and Diseases | Dach I.,University of Aarhus | Olesen C.,Center for Membrane Pumps in Cells and Diseases | Olesen C.,University of Aarhus | And 10 more authors.
Journal of Biological Chemistry | Year: 2012

The H+,K+-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood.Wedescribe here how pure and functionally active pig gastric H +,K+-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C 12E8, followed by exchange of C12E8 with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H+,K +-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9 -1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α2,β2 dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H+,K+-ATPase. Thus, α,β H+,K +-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca2 +-ATPase and Na+,K+-ATPase. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Dubois C.,French Institute of Health and Medical Research | Dubois C.,Center for Membrane Pumps in Cells and Diseases | Vanden Abeele F.,French Institute of Health and Medical Research | Sehgal P.,Center for Membrane Pumps in Cells and Diseases | And 8 more authors.
FEBS Journal | Year: 2013

The inhibition of sarcoplasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin (Tg) and Tg-type analogues is considered to trigger cell death by activation of apoptotic pathways. Some of these analogues may be useful as antineoplastic agents after appropriate targeting as peptide conjugated prodrugs to cancer cells. With this in mind, this study evaluates the effect on LNCaP androgen-sensitive cancer cells of thapsigargin substituted with 12-aminododecanoyl linkers and Leu (Leu-8ADT), aspartate (Asp-8ADT) or Boc-8ADT. Our results show that both Leu-8ADT and Asp-8ADT result in rapid ER calcium depletion and an influx of calcium across the plasma membrane by activation of store-operated calcium entry. By contrast, ER Ca2+ depletion by Boc-8ADT is a very slow process that does not perceptibly increase cytosolic Ca2+ and activate store-operated calcium entry, because the inhibition of SERCA with this compound is very slow. Nevertheless, we find that Boc-8ADT is a more efficient inducer of apoptosis than both Tg and Leu-8ADT. Compared with Tg and the other analogues, apoptosis induced by Asp-8ADT is very modest, although this compound also activates store-operated calcium entry and at high concentrations (1 μm) causes severe morphological changes, reflecting decreased cell viability. We conclude that many factors need to be considered for optimization of these compounds in antineoplastic drug design. Among these ER stress induced by Ca2+ endoplasmic reticulum mobilization seems particularly important, whereas the early cytosolic increase of Ca2+ concentration preceding the executive phase of apoptosis appears to be of no, or little, consequence for a subsequent apoptotic effect. Inhibition of sarcoplasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin (Tg) and Tg analogues is considered to trigger cell death by activation of calcium mediated apoptotic pathways. The present study evaluates the effect of thapsigargin analogues on prostate cancer cells and among a number of surprising findings demonstrate that an early cytosolic increase of Ca2+ concentration is not involved in apoptosis induction. © 2013 FEBS.

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