Измерение<em> In Vitro</em> АТФазы для ферментативного определения характеристик
Summary
We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.
Abstract
Аденозинтрифосфата-гидролизом ферменты или АТФазы, играют решающую роль в разнообразных клеточных функций. Эти динамические белки могут вырабатывать энергию для механической работы, такими как незаконный оборот белка и деградации, растворенной транспорта и клеточных движений. Протокол , описанный здесь , является основным анализом для измерения активности в пробирке очищенных ATPases для функциональной характеристике. Белки гидролизуются АТФ в реакции, что приводит к неорганическим высвобождение фосфата, и количество фосфата освободила затем количественно с помощью колориметрического анализа. Это хорошо адаптируется протокол может быть настроен для измерения активности АТФазы в кинетических или конечных точек анализов. Представитель протокола приводится здесь основывается на деятельности и требований белка EpsE, ААА + АТФазы участвуют в II типа секрецией бактерии холерных вибрионов. Количество очищенного белка, необходимого для измерения активности, продолжительность анализа и выбор времени и количество SAmpling интервалы, буфер и солевой состав, температура, кофакторы, стимуляторы (если таковые имеются) и т.д. могут отличаться от тех , которые описаны здесь, и , таким образом , некоторые оптимизации могут быть необходимы. Этот протокол обеспечивает базовую основу для характеризации АТФазы и может быть выполнена быстро и легко отрегулировано по мере необходимости.
Introduction
ATPases are integral enzymes in many processes across all kingdoms of life. ATPases act as molecular motors that use the energy of ATP hydrolysis to power such diverse reactions as protein trafficking, unfolding, and assembly; replication and transcription; cellular metabolism; muscle movement; cell motility; and ion pumping1-3. Some ATPases are transmembrane proteins involved in transporting solutes across membranes, others are cytoplasmic and may be associated with a biological membrane such as the plasma membrane or those of organelles.
AAA+ ATPases (ATPases associated with various cellular activities) make up a large group of ATPases that share some sequence and structural conservation. These proteins contain conserved nucleotide binding motifs such as Walker-A and -B boxes and form oligomers (generally hexamers) in their active state1. Large conformational changes in these proteins upon nucleotide binding have been characterized among diverse members of the AAA+ family. EpsE is a AAA+ ATPase and member of the bacterial Type II/IV secretion subfamily of NTPases4-6. EpsE powers Type II Secretion (T2S) in Vibrio cholerae, the causative agent of cholera. The T2S system is responsible for the secretion of a wide variety of proteins, such as the virulence factor cholera toxin that causes profuse watery diarrhea when V. cholerae colonizes the human small intestine7.
Techniques for quantitating in vitro ATPase activity are varied, but commonly measure phosphate release using colorimetric, fluorescent, or radioactive substrates8-11. We describe a basic method for determining in vitro ATPase activity of purified proteins using a colorimetric assay based on a commercially available malachite green-containing substrate that measures liberated inorganic phosphate (Pi). At low pH, malachite green molybdate forms a complex with Pi and the level of complex formation can be measured at 650 nm. This simple and sensitive assay may be used to functionally characterize new ATPases and to evaluate the roles of potential activators or inhibitors, to determine the importance of domains and/or specific residues, or to assess the effect of particular treatments on enzymatic activity.
Protocol
Representative Results
Discussion
Это общий протокол для измерения экстракорпоральное АТФазы активность очищенных белков для биохимических характеристик. Этот метод легко оптимизированы; например, регулируя количество белка, буфера и солевых композиций, температуры и изменения длины анализа и интервалы времен?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).
Materials
| HEPES buffer | Fisher | BP310-500 | |
| Sodium chloride | Fisher | BP358-212 | |
| Magnesium chloride | Fisher | BP214-500 | |
| Adenosine triphosphate (ATP) | Fisher | BP41325 | |
| 96-well plates (clear, flat-bottom) | VWR | 82050-760 | |
| BIOMOL Green | Enzo Life Sciences | BML-AK111 | Preferred phosphate detection reagent. Caution: irritant. |
| Microplate reader | BioTek Synergy or comparable | ||
| Prism 5 | GraphPad Software | ||
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