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Development of a new and reliable assay for choline kinase using 31P NMR
- Source :
- Heliyon, Vol 5, Iss 10, Pp e02585-(2019)
- Publication Year :
- 2019
- Publisher :
- Elsevier BV, 2019.
-
Abstract
- Choline kinase catalyzes the conversion of choline to phosphocholine (PC) by transferring a phosphate group from adenosine triphosphate (ATP) as the first step in the biosynthetic pathway for the membrane phospholipid phosphatidylcholine, an essential pathway in the Leishmania parasitic protozoan. Commonly used methods for kinetically quantifying the enzyme include a radioisotope assay utilizing labeled choline and a coupled spectrophotometric assay with multiple enzymes and substrates that indirectly measures choline kinase activity. When testing potential inhibitors with the coupled assay, results can cast doubt on whether choline kinase is being inhibited or one of the coupled enzymes. Therefore, 31P NMR spectroscopy was used to quantitatively measure the formation of the key product, phosphocholine, and to evaluate choline kinase activity. Interrogation of 31P NMR spectroscopy offers a number of benefits. Since this isotope is 100% abundant and has a relatively large gyromagnetic ratio, it is considered one of the more sensitive nuclides. As such, the need for costly isotopic enriched phosphorous is not required and detection of the 31P signal is possible even at relatively low concentrations. The enzymatic activity of Leishmania infantum choline kinase was able to be directly measured via integration of the 31P resonance associated with the phosphocholine product (δ = 3.94 ppm). These initial studies reveal that a 31P NMR spectroscopic-based assay could be used for testing substrate or transition state analogs as competitive inhibitors of Leishmania choline kinase that may prevent phosphatidylcholine synthesis in the parasite.
- Subjects :
- 0301 basic medicine
Kinase
Choline kinase
Phospholipid
TBC
Choline
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Transition state analog
Phosphatidylcholine
31P NMR
lcsh:Social sciences (General)
lcsh:Science (General)
Leishmaniasis
Phosphocholine
Multidisciplinary
030104 developmental biology
chemistry
Biochemistry
Enzyme
lcsh:H1-99
Adenosine triphosphate
030217 neurology & neurosurgery
lcsh:Q1-390
Subjects
Details
- ISSN :
- 24058440
- Volume :
- 5
- Database :
- OpenAIRE
- Journal :
- Heliyon
- Accession number :
- edsair.doi.dedup.....1b09ef4e97fdec3e2e046c12980f3fba
- Full Text :
- https://doi.org/10.1016/j.heliyon.2019.e02585