Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors.
J Vis Exp
; (153)2019 11 23.
Article
in En
| MEDLINE
| ID: mdl-31814619
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Pyrophosphatases
/
Thermotoga maritima
/
Drug Evaluation, Preclinical
/
Enzyme Inhibitors
/
Anti-Bacterial Agents
Type of study:
Diagnostic_studies
/
Screening_studies
Language:
En
Journal:
J Vis Exp
Year:
2019
Document type:
Article