Bioinformatics, enzymologic properties, and comprehensive tracking of Plasmodium falciparum nucleoside diphosphate kinase.
ABSTRACT The gene encoding for nucleoside diphosphate kinase from Plasmodium falciparum was obtained by polymerase chain reaction (PCR) and expressed in Escherichia coli. Tracking kinases is strenuous work due to many functional and technical deficits. Tracking of Plasmodium falciparum nucleoside diphosphate kinase (PfNDK) was carried out by conventional enzyme assays combined by isothermal titration calorimetry (ITC). ITC proved an efficient tracking method with rapid, accurate, and confident target confirmation. In addition, it provides substrate affinity and full thermodynamic profile in one experiment. Magnesium ions were found to be essential for nucleoside diphosphate (NDP) kinase activity; however, the absence of Mg(2+) did not completely interfere with the binding of nucleotides. The substrate recognition was found to depend on enthalpic forces with little entropic contributions. However, in the absence of magnesium ions the nucleotides actively bind to the enzyme driven by hydrophobic forces. The enzyme showed specific activity that was within the average of known enzymes; however, it was at least two-fold higher than that of the human enzyme.
- Journal of Biological Chemistry 12/1966; 241(21):4917-22. · 4.65 Impact Factor
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ABSTRACT: The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstrate a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.Protein Science 08/2008; 17(10):1798-804. · 2.74 Impact Factor
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ABSTRACT: The information available after decoding the genome of the human species and many others is opening the possibility of new approaches to target thousands of protein interactions critical for a continuously increasing list of genetic and infectious diseases and pathologies, and to understand complex regulatory pathways and interaction networks describing cell function and interrelation. There is a need for a reliable technique offering the capability of measuring accurately macromolecular interactions (e.g. protein/ligand, protein/protein, protein/nucleic acid) in the laboratory. Compared to other analytical techniques, isothermal titration calorimetry (ITC) exhibits some important advantages for characterizing intermolecular interactions and binding equilibria. ITC is suitable for characterizing both low affinity interactions (e.g. protein network regulation and natural ligands) and high affinity interactions (e.g. rational drug design). Considering the advanced technological level reached as well as the outstanding quality of the information accessible through this technique, ITC is expected to play a very prominent role in the next years in the areas of rational drug design and protein network regulation.Biophysical Chemistry 05/2005; 115(2-3):115-24. · 2.28 Impact Factor