Vidilaseris, K, Johansson, NG, Turku, A et al. (5 more authors) (2019) Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors. Journal of Visualized Experiments, 153. e60619. ISSN 1940-087X
Abstract
Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 Journal of Visualized Experiments. This is an author produced version of a paper published in Journal of Visualized Experiments. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Biochemistry; Issue 153; membrane-bound pyrophosphatase; Thermotoga maritima; inhibitor screening; molybdenum blue reaction; protist diseases; drug design |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 29 Jan 2020 14:40 |
Last Modified: | 23 May 2020 00:38 |
Status: | Published |
Publisher: | Journal of Visualized Experiments (JoVE) |
Identification Number: | 10.3791/60619 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:156192 |