Conductimetric assay of pyroglutamyl peptidase activity

Besson, Christine; Vessillier, Sandrine; Gonzales, Thierry; Saulnier, Joëlle; Wallach, Jean

doi:10.1016/0003-2670(94)80314-5

Cited by 6 publications

(5 citation statements)

References 12 publications

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“…Conductimetric assays have a multitude of potential uses and provide a method of choice for very many enzymes (12,17,19,28). However one enzyme for which this assay method is outstandingly suitable is inorganic pyrophosphatase, where the extension to coupled detection methods has wide ramifications.…”

Section: Discussionmentioning

confidence: 99%

A Direct Pyrophosphatase-coupled Assay Provides New Insights into the Activation of the Secreted Adenylate Cyclase from Bordetella pertussis by Calmodulin

Lawrence¹,

Coote²,

Kazi³

et al. 2002

Journal of Biological Chemistry

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Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC 4.6.1.1) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k on by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated.The secreted adenylate cyclase (CyaA) 1 of Bordetella pertussis is an important virulence factor and has a number of interesting structural and kinetic properties (1, 2). The N-terminal domain, containing the catalytic and calmodulin-binding domains, is joined to a large RTX (repeats in toxin) domain for membrane translocation (3, 4). Immunological evidence indicates that the catalytic domain may be of eukaryotic origin (5), but the calmodulin binding domain is bipartite and does not resemble classical calmodulin target sites very closely (6). CyaA, and the related edema factor from Bacillus anthracis, are among the most active adenylate cyclases yet characterized and show the largest known responses to calmodulin (4,7,8). CyaA is therefore ideal for evaluating new strategies for assay of adenylate cyclase and calmodulin and calmodulin antagonists (9). The fact that the catalytic reaction generates pyrophosphate as a second product has attracted little attention, but offers the possibility of assay by coupled detection of pyrophosphate cleavage; this could, in principle, enable continuous assays to be devised for many other important enzymes.Radiochemical assays for nucleotide cyclases (10, 11) tend to be used as single time point determinations, and due to logistics of isotope use, especially for 32 P, there is a tendency to run assays in large batches on collected samples. The aim here was to set up a simple rapid continuous recording method based on conductimetric determination of pyrophosphate cleavage (12) to be readily available for quality control and routine monitoring and which might be useful for assaying calmodulin and its inhibitors. However, the first application of the new method revealed time-dependent features of the kinetics for reactions initiated by addition of calmodulin and drew attention to proble...

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Section: Discussionmentioning

confidence: 99%

A Direct Pyrophosphatase-coupled Assay Provides New Insights into the Activation of the Secreted Adenylate Cyclase from Bordetella pertussis by Calmodulin

Lawrence¹,

Coote²,

Kazi³

et al. 2002

Journal of Biological Chemistry

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“…Various materials have been tested: platinum [5, 15-22], gold [5, 11, 14, 18, 23-25], aluminium [11, 14, 26], nickel [11, 14, 26], copper [11, 14, 21], titanium [5, 11, 14], chromium [11, 14], Ta 2 O 5 [12, 13], silver [22] and carbon [27]. In general, all these materials are suitable, especially when high-frequency ac is used.…”

Section: Transducers For Conductometric Biosensorsmentioning

confidence: 99%

“…One important aspect in the development of conductometric interdigital transducers is the proper selection of electrode material. Various materials have been tested: platinum [ 5 , 15 - 22 ], gold [ 5 , 11 , 14 , 18 , 23 - 25 ], aluminium [ 11 , 14 , 26 ], nickel [ 11 , 14 , 26 ], copper [ 11 , 14 , 21 ], titanium [ 5 , 11 , 14 ], chromium [ 11 , 14 ], Ta 2 O 5 [ 12 , 13 ], silver [ 22 ] and carbon [ 27 ]. In general, all these materials are suitable, especially when high-frequency ac is used.…”

Section: Transducers For Conductometric Biosensorsmentioning

confidence: 99%

Conductometric Microbiosensors for Environmental Monitoring

Jaffrézic‐Renault

Dzyadevych

2008

Sensors

211

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This review presents the principles of conductometric measurements in ionic media and the equivalent electrical circuits of different designs for conductometric measurements. These types of measurements were first applied for monitoring biocatalytic reactions. The use of conductometric microtransducers is then presented and detailed in the case of pollutant detection for environmental monitoring. Conductometric biosensors have advantages over other types of transducers: they can be produced through inexpensive thin- film standard technology, no reference electrode is needed and differential mode measurements allow cancellation of a lot of interferences. The specifications obtained for the detection of different pesticides, herbicides and heavy metal ions, based on enzyme inhibition, are presented as well as those obtained for the detection of formaldehyde, 4- chlorophenol, nitrate and proteins as markers of dissolved organic carbon based on enzymatic microbiosensors.

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“…Наступним важливим чинником успіху розроб ки кондуктометричних гребінчастих перетворюва чів є матеріал електродів. При розробці використо вували різноманітні матеріали: платину [41,[47][48][49][50][51][52][53][54], золото [19,41,43,46,50,55,56], алюміній [43,46,57], нікель [43,46,57], мідь [43,46,53], титан [41,43,46], хром [43,46], Та 2 0 5 [44,45], срібло [54 ] і вуглець [66 ]. Взагалі усі перераховані матеріали можна застосовувати, особливо якщо роботу виконують при змінному струмі високої частоти.…”

Section: перетворювачі для кондуктометричних біо-unclassified

“…Кондуктометричний метод було застосовано і для вивчення піроглутамілпептидази, при цьому отримано константи Міхаеліса, які відповідали 0,34 і 0,47 мМ для похідних аланіну і тирозину від повідно [66].…”

Section: перетворювачі для кондуктометричних біо-unclassified

Untitled

2005

Biopolym. Cell

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Розглянуто теоретичні основи кондуктометричного методу вимірювань та можливість його використання для реєстрації перебігу ферментативних процесів, що проілюстровано відповідними даними різних дослідників. Наведено приклади багатьох кондуктометричних перетворювачів, які використовують при створенні біосенсорів, засоби іммобілізації активного матеріалу на їхню поверхню та схеми і методики вимірювань. Описано деякі моделі кондуктометричних фермент них біосенсорів, обговорено їхні переваги і недоліки, а також перспективи подальшого розвитку.

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Conductimetric assay of pyroglutamyl peptidase activity

Cited by 6 publications

References 12 publications

A Direct Pyrophosphatase-coupled Assay Provides New Insights into the Activation of the Secreted Adenylate Cyclase from Bordetella pertussis by Calmodulin

A Direct Pyrophosphatase-coupled Assay Provides New Insights into the Activation of the Secreted Adenylate Cyclase from Bordetella pertussis by Calmodulin

Conductometric Microbiosensors for Environmental Monitoring

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