Affinity labeling of the (Na+ + Mg2+)-ATPase from A choleplasma laidlawii B membranes by the 2′,3′-dialdehyde derivative of adenosine 5′-triphosphate

George, Rajan; McElhaney, Ronald N.

doi:10.1016/0005-2736(85)90229-9

Cited by 16 publications

(4 citation statements)

References 19 publications

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“…Because o-ATP prevented formation of the SDS-resistant PA oligomer, we hypothesized that o-ATP may alter endosomal pH, possibly through effects on the vacuolar ATPase. It has been shown that o-ATP modifies and inhibits many ATPases (13,25,26,39,46,52). We tested the effects of o-ATP on endosomal pH directly by using two pH-sensitive dyes, acridine orange and LysoTracker Red.…”

Section: Resultsmentioning

confidence: 99%

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Oxidized ATP Protection against Anthrax Lethal Toxin

et al. 2006

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Bacillus anthracis lethal toxin (LT) induces rapid lysis (<90 min) of murine macrophages from certain inbred strains. The mechanism for LT-induced cytolysis is currently unknown. We hypothesized that the ATP-activated macrophage P2X7 receptors implicated in nucleotide-mediated macrophage lysis could play a role in LT-mediated cytolysis and discovered that a potent P2X7 antagonist, oxidized ATP (o-ATP), protects macrophages against LT. Other P2X7 receptor antagonists, however, had no effect on LT function, while oxidized nucleotides, o-ADP, o-GTP, and o-ITP, which did not act as receptor ligands, provided protection. Cleavage of the LT substrates, the mitogen-activated protein kinases, was inhibited by o-ATP in RAW274.6 macrophages and CHO cells. We investigated the various steps in the intoxication pathway and found that binding of the protective-antigen (PA) component of LT to cells and the enzymatic proteolytic ability of the lethal factor (LF) component of LT were unaffected by o-ATP. Instead, the drug inhibited formation of the sodium dodecyl sulfate-resistant PA oligomer, which occurs in acidified endosomes, but did not prevent cell surface PA oligomerization, as evidenced by binding and translocation of LF to a protease-resistant intracellular location. We found that o-ATP also protected cells from anthrax edema toxin and diphtheria toxin, which also require an acidic environment for escape from endosomes. Confocal microscopy using pH-sensitive fluorescent dyes showed that o-ATP increased endosomal pH. Finally, BALB/cJ mice injected with o-ATP and LT were completely protected against lethality.Anthrax toxin consists of three polypeptides that form two binary toxins. The lethal toxin (LT) and edema toxin (ET) each contain the receptor binding protein, protective antigen (PA), combined with an enzymatic cargo protein. Lethal factor (LF), a protease which cleaves members of the mitogen-activated protein kinase family (MEKs), and edema factor (EF), a calmodulin-dependent adenylate cyclase, are transported into the cell cytoplasm by PA oligomers. These oligomers can form only after binding and cleavage of PA from an 83-kDa protein (PA83) to its 63-kDa form (PA63) by furin at the cell surface. The heptameric PA63 oligomers carrying LF and/or EF are then conformationally altered in an intracellular acidic compartment to allow passage of their cargo to the cytosol through a pore (for a review, see reference 9).LT is present at high concentrations in the blood of infected animals and is sufficient to induce some symptoms of anthrax and lethality in animal models (4,21,41,11,29,30,56). Although many LT-mediated effects in cells and animals have been described over the years, the actual mechanism by which LT causes cell or animal death is unknown. Studying the toxin's unique induction of rapid lysis in murine macrophage lines (such as RAW264.7 and J774.A1 cells) or in primary macrophages from BALB/cJ mice may provide a better understanding of its function and cellular targets (23). The macrophage lysis assay also pro...

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Section: Resultsmentioning

confidence: 99%

“…These results have raised new questions about when o-ATP can reliably be used as a P2X7 receptor blocker and how it exerts its P2X7-independent effects (14). Beigi et al (5) (25), and Escherichia coli GroEL (52). Modification was often accompanied by loss of function.…”

Section: Discussionmentioning

confidence: 99%

Oxidized ATP Protection against Anthrax Lethal Toxin

et al. 2006

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“…As to the affinity tag we elected to use either the azido functional group, which is stable in dark but photolyses to a highly reactive N Å -adenine radical on exposure to UV [14][15][16] or a ring-opened ribose-dialdehyde, which has been shown to spontaneously form stable Schiff base-like moieties with primary amines in proteins. [17][18][19] The route to dial affinity probe 1 was relatively simple (see Scheme 1); 20 the synthesis of mant-Ap 4 A has been reported previously 4 and conversion to the dialdehyde was effected by the means of sodium periodate oxidation. 17 The main obstacles were the purification and handling of the final product owing to the ribose-dialdehyde reactivity.…”

Section: To Detect and Identify Intracellular Ap 4 A-binding Pro-mentioning

confidence: 99%

“…[14][15][16][17][18][19] Fluorescence emission spectra from each protein-containing mixture are shown (Fig. 2a).…”

Section: To Detect and Identify Intracellular Ap 4 A-binding Pro-mentioning

confidence: 99%