計測<em>インビトロ</em酵素特性評価のための> ATPase活性
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
アデノシン三リン酸加水分解酵素、またはATPアーゼは、細胞機能の多様な配列に重要な役割を果たしています。これらの動的なタンパク質は、タンパク質輸送および分解、溶質輸送、および細胞の動きとして、機械的な作業のためのエネルギーを生成することができます。ここで説明するプロトコルは、機能的な特徴付けのために精製されたATPアーゼのインビトロ活性を測定するための基本的なアッセイです。タンパク質は、無機リン酸の放出をもたらす反応でATPを加水分解し、遊離したリン酸塩の量を、比色アッセイを用いて定量されます。この高度に適応プロトコルは、運動またはエンドポイントアッセイにおいてATPアーゼ活性を測定するために調整することができます。代表的なプロトコルは活動とEPSEの要件に基づいて、ここで提供され、AAA + ATPアーゼは、細菌コレラ菌にタイプIIの分泌に関与します。活性を測定するために必要な精製されたタンパク質の量は、アッセイの長さおよびSAのタイミングおよび数mpling間隔は、緩衝液および塩組成、温度、補因子、刺激剤（もしあれば）、 等は、ここで説明したものと異なる場合があり、したがって、いくつかの最適化が必要であってもよいです。このプロトコルは、特徴付けるATPアーゼのための基本的な枠組みを提供し、迅速に行われ、容易に、必要に応じて調整することができます。
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
これは、生化学的な特徴付けのために精製されたタンパク質のin vitroでのATPase活性に測定するための一般的なプロトコルです。この方法は、容易に最適化されています。例えば、蛋白質、緩衝液および塩組成物の量、温度を調整し、アッセイ長さ（間隔の合計数を増やすなど）の間隔を変化させると、活性定量を改善することができます。市販のマラカイトグリーンベースの試?…
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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