Elucidation of Mg²⁺ binding activity of adenylate kinase from Mycobacterium tuberculosisH₃₇Rv using fluorescence studies

Abstract

Adenylate kinase (AK) is a small ubiquitous enzyme that catalyzes the reversible transfer of the terminal phosphate group from adenine triphosphate (ATP): magnesium ion (Mg²⁺) to adenine monophosphate (AMP) to form two molecules of adenine diphosphate (ADP). AK thus maintains the homeostasis of adenine nucleotides in eukaryotes and prokaryotes. Because the [ATP]/[ADP] ratio is an important parameter in energy regulation in cells, Mg²⁺-activated AK has an important biological role, particularly in the case of bacteria, as imbalance in the ratio of [ATP]/[ADP] has been associated with alteration in its DNA supercoiling state. In the present study, magnesium-binding assays were carried out by systematically varying the concentrations of Mg²⁺, protein, AMP, ATP, and indicator in kinetic experiments. We report evidence that during magnesium-binding assay, the fluorescence level of the indicator "Mag-Indo-1" changes with protein concentration, suggesting that magnesium ions are binding to AK. The dual activity of AK both as nucleoside monophosphate and diphosphate kinases suggests that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism. According to the proposed model, the magnesium-activated AK exhibits an increase in its forward reaction rate compared with the inactivated form. These findings imply that Mg²⁺ could be an important regulator in the energy signaling network in cells.

Cloning and characterization of a novel PE_PGRS60 protein (Rv3652) of Mycobacterium tuberculosis H37Rv exhibit fibronectin‐binding property

“…In the past, protein purification methods were described in various studies ; modified purification methods are described here briefly, in which the cell pellet was resuspended in 20 mL sonication buffer (50 mM Tris–Cl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/mL aprotonin). The cells were sonicated on ice for 10 Min at 10 or 20 Sec off pulse.…”

Fluorescent probes for the detection of magnesium ions (Mg2+): from design to application

“…The K d of Mag-indo-1 for Mg 2+ is lower than that of Mag-fura-2 (the parameter is shown in Table 1), which is conducive for the measurement of high spikes in intracellular Mg 2+ , for example the temporal analysis of Ca 2+induced Mg 2+ mobilization in neurons or the glutamatestimulated Mg 2+ concentration change. [37][38][39] Later, several other uorescent probes which possess the APTRA group as the binding site for Mg 2+ have been developed, such as Mag-uo-4 and Magnesium Green. 40,41 The remarkable feature of those probes compared to the aforementioned ones is that they are excited by visible light (see Table 1) rather than UV irradiation which is regarded to be more cytotoxic.…”

Development of Functional Fluorescent Molecular Probes for the Detection of Biological Substances

“…Mag-fura-2 undergoes an appreciable shift in excitation wavelength upon binding to Mg 2+ , whereas mag-indo-1 shows a shift in both its excitation and emission wavelengths. The affinity of mag-indo-1 for Mg 2+ is lower than that of mag-fura-2, which is useful for the measurement of high spikes in intracellular Mg 2+ , such as the glutamate-stimulated Mg 2+ concentration change or the temporal analysis of Ca 2+ -induced Mg 2+ mobilization in neurons [ 41 , 42 , 43 ].…”