An improved assay for nanomole amounts of inorganic phosphate

Lanzetta, Peter A.; Alvarez, Lawrence J.; Reinach, Peter S.; Candia, Oscar A.

doi:10.1016/0003-2697(79)90115-5

Cited by 2,080 publications

(1,397 citation statements)

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“…Electrochemical, AFM, and QCM measurements were carried out as reported previously 16a, 17, 27. Inorganic phosphate (P i ) determination was performed by the Green Malachite assay 28. ATP concentration was measured using the Luciferase assay kit from Molecular Probes..…”

Section: Methodsmentioning

confidence: 99%

H₂‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

et al. 2016

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ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0‐ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode‐assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.

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Section: Methodsmentioning

confidence: 99%

H₂‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

et al. 2016

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“…Incubation times and protein concentrations were chosen to ensure the linearity of the enzymatic reaction. The release of inorganic Pi was measured with the malachite green method [30]. KH 2 PO 4 was used as a Pi standard.…”

Section: Artificial Postmortem Delaymentioning

confidence: 99%

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

Aliagas

Villar-Menéndez

Sévigny

et al. 2013

Purinergic Signalling

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Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D 2 receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5′-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n=13) compared with aged-matched controls (n=10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ectonucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5′-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.

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“…ATPase activity assays were performed with a slight modifi cation according to the malachite green method [36,37]. Unless stated otherwise, all reactions were performed at 40°C in the following conditions (standard assay conditions).…”

Section: Atpase Activity Assaymentioning

confidence: 99%

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

et al. 2009

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The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purifi ed to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purifi ed BlDnaK was 40°C in the presence of 100 mM KCl. The purifi ed BlDnaK had a V max of 32.5 nmol Pi/min and a K M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg 2+ and K + ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.

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An improved assay for nanomole amounts of inorganic phosphate

Cited by 2,080 publications

References 4 publications

H₂‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

H₂‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

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An improved assay for nanomole amounts of inorganic phosphate

Cited by 2,080 publications

References 4 publications

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

Contact Info

Product

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H₂‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

H₂‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration