Medindo<em> In Vitro</em> Actividade ATPase para enzimática Caracterização
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
enzimas de hidrólise de trifosfato de adenosina, ou ATPases, desempenham um papel crítico num conjunto diversificado de funções celulares. Estas proteínas dinâmicas podem gerar energia para o trabalho mecânico, tais como o tráfico de proteínas e degradação, transporte de soluto e movimentos celulares. O protocolo aqui descrito é um ensaio de base para a medição da actividade in vitro de ATPases purificadas para a caracterização funcional. Proteínas hidrolisar ATP numa reacção que resulta na libertação de fosfato inorgânico, e a quantidade de fosfato libertado é então quantificada utilizando um ensaio colorimétrico. Este protocolo altamente adaptável pode ser ajustado para medir a actividade de ATPase em ensaios cinéticos ou terminal. Um protocolo representante é fornecido aqui com base na atividade e as exigências de Epse, a AAA + ATPase envolvida no Tipo II Secreção na bactéria Vibrio cholerae. A quantidade de proteína purificada necessária para medir a actividade, duração do ensaio e o calendário e número de saintervalos mpling, tampão e a composição de sal, temperatura, co-factores, estimulantes (se houver), etc., podem variar a partir dos descritos aqui, e, assim, alguma optimização pode ser necessário. Este protocolo fornece uma estrutura básica para ATPases que caracterizam e pode ser realizado de forma rápida e facilmente ajustado conforme necessário.
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
Este é um protocolo geral para a medição da actividade ATPase in vitro de proteínas purificadas para a caracterização bioquímica. Este método é facilmente optimizado; Por exemplo, ajustando a quantidade de proteína, tampão e composições de sal, temperatura, e variando-se o comprimento de ensaio e intervalos (incluindo o aumento do número total de intervalos) pode melhorar a actividade de quantificação. reagentes verde à base de malaquita comercialmente disponíveis são altamente sens?…
Divulgations
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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