A rapid method for determining ATP by the firefly luciferin-luciferase system

Neufeld, Harold A.; Towner, Richard D.; Pace, J.

doi:10.1007/bf01922604

Cited by 21 publications

(13 citation statements)

References 9 publications

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“…The ATP present in the medium was estimated with a bioluminescent assay (Neufeld et al, 1975). Fifty microliters of the cellular supernatant was added to a test tube, which was transferred to the chamber of a Lumat LB9507 luminometer (Berthold Technologies, Wildbad, Germany).…”

Section: Methodsmentioning

confidence: 99%

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Modulation by LL-37 of the Responses of Salivary Glands to Purinergic Agonists

Pochet

Tandel²,

Querriére³

et al. 2006

Mol Pharmacol

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The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calciuminsensitive phospholipase A 2 (PLA 2 ). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by, a rather specific inhibitor of P2X 7 receptors in mice. LL-37 also increased [Ca 2ϩ ] i in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca 2ϩ ] i and the activation of phospholipase D by ATP. It potentiated the activation of the PLA 2 by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X 7 receptors but modulates their responses.Cathelicidins are proteins involved in the first phases of our defenses against pathogens. Their isolation relied on the analogy of their N-terminal proregions with cathelin, an inhibitor of cathepsin L originally isolated from bovine leukocytes (Ritonja et al., 1989). This highly conserved proregion (approximately 100 amino acids) shares many similarities with cystatins, inhibitors of the cysteine proteases. The Cterminal domain of cathelicidins varies among species both in terms of length (12-100 amino acids) and structure. The N-and C-terminal domains are separated by a sequence recognized by proteases. The digestion in the extracellular medium of the propeptides by elastase (Panyutich et al., 1997) or proteinase 3 (Sorensen et al., 2001) releases the C-terminal peptides originally described as antimicrobial but that now prove to be more immunomodulatory than antimicrobial in physiological conditions (Bowdish et al., 2005). Most of the studies on cathelicidins have focused on the C-terminal peptides. These peptides are ubiquitous. They are secreted by macrophages, lymphocytes, epithelial cells, keratinocytes, cells lining the upper respiratory tree, and vaginal cells (Bals and Wilson, 2003). LL-37, the only peptide from human origin, is derived from an antibacterial protein of 18 kDa. LL-37 and its only analog in mouse, the cathelinrelated antimicrobial peptide, are cationic and transform from a random coil in aqueous solution to an amphipathic ␣-helix at the contact of a membrane (Yeaman and Yount, 2003). The peptide binds to the bacteria by electrostatic interactions between the positive charges on one side of its

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Section: Methodsmentioning

confidence: 99%

“…The cells were incubated with 10 M LL-37. After centrifugation, the ATP content of the medium was estimated with a luciferin-luciferase mixture (Neufeld et al, 1975). Cells were incubated in a test tube in the chamber of the luminometer and preincubated with the ATP assay mix before the addition of LL-37 (Fig.…”

Section: Ll-37 and Salivary Glands 2041mentioning

confidence: 99%

Modulation by LL-37 of the Responses of Salivary Glands to Purinergic Agonists

Pochet

Tandel²,

Querriére³

et al. 2006

Mol Pharmacol

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“…The reaction mixture was incubated at room temperature for 5 min, and the reaction was terminated with 0.1 ml of 12% (vol/vol) HClO 4 . ATP levels were estimated by using a luciferinluciferase bioluminescence assay (19). ATP solutions of known concentrations were used as standards.…”

Section: Methodsmentioning

confidence: 99%

Dual mode of energy coupling by the oxyanion-translocating ArsB protein

1995

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The arsA and arsB genes of the ars operon of R-factor R773 confer arsenite resistance in Escherichia coli by coding for an anion-translocating ATPase. Arsenite resistance and the in vivo energetics of arsenite transport were compared in cells expressing the arsA and arsB genes and those expressing just the arsB gene. Cells expressing the arsB gene exhibited intermediate arsenite resistance compared with cells expressing both the arsA and arsB genes. Both types of cells exhibited energy-dependent arsenite exclusion. Exclusion of 73 AsO 2 ؊ from cells expressing only the arsB gene was coupled to electrochemical energy, while in cells expressing both genes, transport was coupled to chemical energy, most likely ATP. These results suggest that the Ars anion transport system can be either an obligatory ATP-coupled primary pump or a secondary carrier coupled to the proton motive force, depending on the subunit composition of the transport complex.Bacterial resistance to arsenical and antimonial compounds results from active extrusion of the toxic oxyanions from the resistant cells (15). Efflux of arsenite from oxyanion resistant Escherichia coli cells is catalyzed by a pump whose genes are encoded on the conjugative R factor R773 (5, 10). This aniontranslocating ATPase is composed of two types of polypeptides subunits, the ArsA and ArsB proteins. The 63-kDa ArsA protein is an anion-stimulated ATPase (11, 23) that functions as the catalytic component of the pump and remains attached to the inner membrane through interaction with the ArsB subunit (7, 30). The ArsB polypeptide is a 45-kDa inner membrane protein (26) that presumably forms the anion-conducting pathway.In vivo chemical energy was shown to be both necessary and sufficient for the pump, while electrochemical energy was neither (18,22). These studies were performed with an unc strain of E. coli defective in the H ϩ -translocating ATPase that catalyzes the equilibrium between ATP and the electrochemical proton gradient. This allows the establishment of intracellular conditions such that either chemical energy or electrochemical energy is available for transport (2). While the identity of the chemical used for energy coupling cannot be determined from the in vivo results, the energy requirement in vitro was similarly linked to chemical energy. In everted membrane vesicles, ATP was specifically required for uptake of arsenite, while electrochemical energy was neither necessary nor sufficient (6). Thus, the R773 ArsA-ArsB complex is clearly an obligatorily ATPcoupled pump.Similar arsenite resistance operons have been identified in plasmids of the gram-positive bacteria Staphylococcus aureus(pI258) (12) and Staphylococcus xylosus(pSX267) (24). The staphylococcal ars operons do not contain an arsA gene, yet the resistant cells still actively extrude arsenite (27,28), suggesting that the ArsB protein can transport arsenite in the absence of an arsA gene product. It is possible that the cells use a chromosomally encoded ATPase as a replacement for the ArsA protein or t...

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“…Cell ATP content Intracellular ATP was measured luminometrically employing the luciferase-catalyzed ATP dependent oxidation of luciferin [27]. After carefully removing the culture supernatant, cells were covered with 50 ll ATP-assay solution (buffer: 25 mM HEPES, 5 mM MgSO 4 , 140 mM NaCl, pH 7.8; 5 lg/ml luciferase (Quantilum, Promega) and 10% (v/v) of 10 9 cell ATP releasing reagent (FL-SAR, Sigma).…”

Section: Detection Of Apoptosis By Annexin V-fitc Stainingmentioning

confidence: 99%

Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9 cell cultures

Yang

Dong

et al. 2009

Virus Genes

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The non-structural protein NS1 of Periplaneta fuliginosa densovirus (PfDNV) is a multifunctional protein that has previously been shown to possess ATP-binding, ATPase, site-specific DNA-binding, helicase, and transcription activation activities. We report here an investigation of the cytopathogenicity of this viral non-structural (NS) protein, as well as other two NSs, NS2, and NS3, in cultured insect cells. The expression of NS1 alone potently inhibited cellular gene expression, whereas NS2 and NS3 did not produce a similar effect. The inhibition of gene expression by NS1 was confirmed to be specific and not a simple manifestation of toxicity. For example, NS1 inhibited expression of several reporter genes under the control of different RNA polymerase II promoters, whereas it did not inhibit expression from a T7 RNA polymerase promoter construct. Mapping analysis identified the carboxy-terminal peptide of this protein as the region important for the inhibition of cellular gene expression, suggesting that this inhibition is independent of its DNA-binding activity. Next, the mutagenesis assay showed that ATP-binding was essential for the unique function of this protein. Furthermore, we found that NS2 and NS3 cooperatively enhanced the NS1-induced transcription inhibition. Co-expression of all the three NS proteins in Sf9 cells also led to necrotic cell death by ATP depletion.

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A rapid method for determining ATP by the firefly luciferin-luciferase system

Cited by 21 publications

References 9 publications

Modulation by LL-37 of the Responses of Salivary Glands to Purinergic Agonists

Modulation by LL-37 of the Responses of Salivary Glands to Purinergic Agonists

Dual mode of energy coupling by the oxyanion-translocating ArsB protein

Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9 cell cultures

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