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Antibiotics 2018, 7(1), 24; https://doi.org/10.3390/antibiotics7010024

Parallel Colorimetric Quantification of Choline and Phosphocholine as a Method for Studying Choline Kinase Activity in Complex Mixtures

Food Microbiology and Biotechnology Laboratory, Food and Nutritional Sciences Program, North Carolina A & T State University, 1601 East Market Street, Greensboro, NC 27411, USA
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Received: 27 February 2018 / Revised: 12 March 2018 / Accepted: 14 March 2018 / Published: 17 March 2018
(This article belongs to the Special Issue Top 35 of Antibiotics Travel Awards 2017)
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Abstract

Choline kinase (Chok) is an enzyme found in eukaryotes and Gram-positive bacteria. Chok catalyzes the production of phosphocholine from choline and ATP. This enzyme has been validated as a drug target in Streptococcus pneumonia, but the role Chok enzymatic activity plays in bacterial cell growth and division is not well understood. Phosphocholine production by Chok and its attenuation by inhibitors in the context of complex samples such as cell extracts can currently be quantified by several methods. These include choline depletion measurements, radioactive methods, mass-spectrometry, and nuclear magnetic resonance. The first does not measure phosphocholine directly, the second requires elaborate safety procedures, and the third and fourth require significant capital investments and technical expertise. For these reasons, a less expensive, higher throughput, more easily accessible assay is needed to facilitate further study in Gram-positive Choks. Here, we present the development of a triiodide/activated charcoal/molybdenum blue system for detecting and quantifying choline and phosphocholine in parallel. We demonstrate that this system can reliably quantify changes in choline and phosphocholine concentrations over time in Chok enzymatic assays using cell extracts as the source of the enzyme. This is an easily accessible, convenient, robust, and economical method for studying Chok activity in complex samples. The triiodide/activated charcoal/molybdenum blue system opens new doors into the study choline kinase in Gram-positive pathogens. View Full-Text
Keywords: choline kinase; S. pneumonia; choline; phosphocholine; colorimetric methods; enzymes; gram-positive bacteria choline kinase; S. pneumonia; choline; phosphocholine; colorimetric methods; enzymes; gram-positive bacteria
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Zimmerman, T.; Ibrahim, S.A. Parallel Colorimetric Quantification of Choline and Phosphocholine as a Method for Studying Choline Kinase Activity in Complex Mixtures. Antibiotics 2018, 7, 24.

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