测量<em>体外</em>的酶学性质ATP酶活性
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
三磷酸腺苷水解酶，或ATP酶，在细胞功能多样化阵列发挥关键作用。这些动态蛋白可用于机械工作，如蛋白质运输和退化，溶质运移和细胞运动产生能量。此处所描述的协议是用于测量纯化ATP酶的体外活性功能表征一个基本化验。蛋白水解的ATP以导致的无机磷酸盐的释放反应，和磷酸盐的释放的量，然后用比色测定法进行定量。这个高度适应协议可以进行调整，以测量在动力学或终点测定ATP酶活性。代表性的协议是基于活性和EPSE要求这里提供时，AAA + ATP酶参与II型分泌在细菌霍乱弧菌 。测量活动所需的纯化的蛋白质的量，测定的长度和sa定时和数量mpling间隔，缓冲液和盐的组合物，温度，辅因子，兴奋剂（如果有的话） 等可以从这里描述的变化，并且因此一些优化可能是必要的。该协议为特征的ATP酶的基本框架，并可以快速进行，方便地根据需要进行调整。
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
这是用于对生化表征纯化的蛋白质的体外 ATP酶活性测定通用协议。该方法很容易进行优化;例如，调整的蛋白质，缓冲液和盐的组合物，温度，以及改变该测定长度和间隔（包括增加的间隔的总数）的量可提高活性定量。市售孔雀石基于绿色试剂都高度敏感，而且可以检测少量游离磷酸的（〜100微升50皮摩尔）。因为该测定的敏感性，这是至关重要的使用一次性塑料器皿，超纯水，缓…
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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