Related Experiment Video

Updated: Jan 31, 2026

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS
06:38

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Published on: February 24, 2021

5.8K

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases

Justine Ferey1, David Da Silva1, Cyril Colas2

  • 1Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067, Orléans, France.

Analytica Chimica Acta
|January 8, 2019
PubMed

Insights

Researchers developed simple, rapid, and sensitive enzyme assays to monitor the three-step phosphorylation of thymidine to thymidine triphosphate. These methods are crucial for evaluating nucleoside analogue drugs used in cancer and viral infection treatments.

Area of Science:

  • Biochemistry
  • Enzymology
  • Pharmacology

Background:

  • Nucleosides and their analogues are vital for treating cancers and viral infections.
  • Therapeutic efficacy relies on conversion to active nucleoside triphosphates via kinase-catalyzed phosphorylation.
  • Developing sensitive enzyme activity tests is essential for drug development.

Purpose of the Study:

  • To monitor the three-step phosphorylation of thymidine to thymidine triphosphate.
  • To characterize free and immobilized kinase activities using kinetic models.
  • To establish rapid and sensitive methods for enzyme kinetic studies and phosphorylation monitoring.

Main Methods:

  • Utilized human thymidine kinase 1 (hTK1), human thymidylate kinase (hTMPK), and human nucleoside diphosphate kinase (hNDPK).
  • Employed Michaelis-Menten kinetics to characterize enzyme activities.
  • Applied Flow Injection Analysis with High-Resolution Mass Spectrometry (FIA-HRMS) and Capillary Electrophoresis with UV detection (CE-UV).

Main Results:

  • Successfully monitored the sequential phosphorylation of thymidine.
  • FIA-HRMS provided sensitive and rapid evaluation of the phosphorylation process.
  • Characterized both free and immobilized enzyme kinetics.

Conclusions:

  • Proposed simple, rapid, efficient, and sensitive methods for enzyme kinetic studies.
  • Demonstrated the utility of FIA-HRMS for monitoring successive phosphorylation.
  • Highlighted the importance of these methods for evaluating nucleoside analogue drugs.
Keywords:
Capillary electrophoresisDrug discoveryFIA-HRMSImmobilized kinasesMichaelis-mentenMulti-step enzymatic cascade phosphorylation

More Related Videos

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

19.4K
Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry08:23

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Published on: March 9, 2018

9.3K

Related Concept Videos

High-Resolution Mass Spectrometry (HRMS)01:15

High-Resolution Mass Spectrometry (HRMS)

The resolution of a mass spectrometer depends on the efficiency of separating ions with different ion masses. The mass of an atom is approximated to the sum of the masses of protons and neutrons inside, considering the masses of protons and neutrons as equal. However, the masses of the proton (1.6726 × 10−24 g) and neutron (1.6749 × 10−24 g) are not truly equal. There is a minor error in the expression of atomic masses relative to the simplest atom of hydrogen. For...
2.5K
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
53.8K
Mass Spectrometry: Complex Analysis01:21

Mass Spectrometry: Complex Analysis

Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
1.8K
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
15.1K
Mass Spectrometry: Overview01:19

Mass Spectrometry: Overview

Mass spectrometry is an analytical technique used to determine the molecular mass and molecular formula of a compound. The basic principle of mass spectrometry is to generate ions from the analyte molecule and measure these ion abundances against their molecular mass. One common type of ionization, known as electron ionization or EI, bombards the analyte molecules in the gas phase with high-energy electron beams. The electron beams displace an electron from the molecule and leave behind a...
8.8K
Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
2.5K