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Adenylate kinase

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Adenylate kinase is an enzyme that catalyzes the reversible transfer of a phosphate group between adenine nucleotides, specifically converting ADP (adenosine diphosphate) to ATP (adenosine triphosphate) and AMP (adenosine monophosphate). This enzyme plays a crucial role in cellular energy metabolism and homeostasis.

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Lab products found in correlation

Adenosine triphosphate (atp), by Merck Group (2 mentions) Phosphoenolpyruvate, by Merck Group (2 mentions) Pyruvate kinase, by Merck Group (2 mentions) L cysteine, by Merck Group (1 mentions) L lysine, by Merck Group (1 mentions) Sodium dithionite, by Thermo Fisher Scientific (1 mentions) L methionine, by Merck Group (1 mentions) Pyridoxal 5 phosphate, by Merck Group (1 mentions) Phosphocreatine, by Merck Group (1 mentions) Phosphocreatine kinase, by Merck Group (1 mentions) Ammonium iron 2 sulfate, by Thermo Fisher Scientific (1 mentions) Atp determination kit, by Merck Group (1 mentions) H2dcfda, by Thermo Fisher Scientific (1 mentions) Carbonyl cyanide m chlorophenyl hydrazone, by Merck Group (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) Phenol red, by Merck Group (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Glycerol, by Merck Group (1 mentions) Potassium chloride (kcl), by Merck Group (1 mentions)

5 protocols using adenylate kinase

1

Enzyme Kinetics of PKAL-1 via Coupled Assay

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The enzyme kinetics of PKAL-1 were determined through an enzyme-coupled spectrophotometric assay35 (link). Each 100 μL assay mixture contained 100 mM Tris buffer pH 7.4, 1 mM dithiothreitol, 10 mM MgCl2, 4 mM ATP, 0.9 mM phosphoenolpyruvate, 0.3 mM NADH, 2.5 U pyruvate kinase, 3.5 U lactate dehydrogenase, 10 U adenylate kinase (Sigma M3003, prepared according to manufacturer’s protocol), 100 mM buffered hydroxylamine, and the tested substrates in DMSO (final volume 2.5%). The kinetic assay was initiated by the addition of an enzyme and run at 22 °C. No activity was detected in the assay when initiating with PKAL-1(K488A). GraphPad Prism was used to calculate the apparent kinetic constants.
Feng L., Gordon M.T., Liu Y., Basso K.B, & Butcher R.A. (2021). Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan. Nature Communications, 12, 4912.
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2

Synthesis and Labeling of Lysine Derivatives

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Adenosine triphosphate (ATP), L-methionine, L-cysteine, L-lysine, pyridoxal 5′-phosphate (PLP), adenylate kinase, phosphocreatine kinase, and phosphocreatine were purchased from Sigma. Uniformly labeled 13C and 15N L-lysine were purchased from Cambridge Isotope Laboratories. D,L-lysine-1,2-13C and D,L-lysine-2-13C were purchased from Aldrich. L-lysine-2,6,6-2H, L-lysine-3,3,4,4,5,5,6,6-2H and D,L-lysine-2-15N were purchased from CDN Isotopes. Sodium dithionite was purchased from Acros Organics and ammonium iron(II) sulfate was purchased from Fisher Chemicals. L-[Methyl-13C]-methionine was purchased from Cambridge Isotope Laboratories. All labeled substrates had a minimum of 99 atom % labeling. L-[U-14C] lysine was obtained from NEN Life Sciences Products. SAM and anSAM were prepared in our laboratory as described in the following sections. All other chemicals and reagents were of the highest purity and used as supplied.
Horitani M., Byer A.S., Shisler K.A., Chandra T., Broderick J.B, & Hoffman B.M. (2015). Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5′-dAdo• “Free Radical” Is Never Free. Journal of the American Chemical Society, 137(22), 7111-7121.
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3

Determination of Adenine Nucleotides via Bioluminescence

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In summary,
bacteria were resuspended in 600 μL of 80 °C absolute ethanol
and incubated in an 80 °C water bath for 10 min. Subsequently,
1.4 mL of buffer solution (composed of 2.5 mL of 1 M Tris, 5 mL of
100 mM MgSO4, 1 mL of 100 mM EDTA, 41.5 mL of ddH2O, and 300 μL of 5 M NaOH) was added, followed by centrifugation
at 12,000 rpm for 2 min. The resulting supernatant was utilized for
the determination of adenine nucleotide through a luciferin–luciferase
bioluminescence assay. To measure ATP, a 10 μL reaction buffer
was added, consisting of 3.75 mL of 1 M Tris, 1.25 mL of 200 mM MgCl2, 6.25 mL of 100 mM KCl, and 37.5 mL of ddH2O,
and adjusted to a pH of 7.75. No ATP-generating enzyme was included
in this buffer. To measure ADP, a reaction buffer containing pyruvate
kinase (Sigma-Aldrich) and phosphoenolpyruvate (Sigma-Aldrich) was
added. To measure AMP, a reaction buffer containing pyruvate kinase,
phosphoenolpyruvate, and adenylate kinase (Sigma-Aldrich) was added.
The quantity of ADP was determined by subtracting the ATP value from
the combined ATP + ADP value, while the quantity of AMP was computed
based on the disparity between the ATP + ADP + AMP content and the
ATP + ADP content.
Li X., Feng D., Zhou J., Wu W., Zheng W., Gan W., Jiang M., Li H., Peng X, & Zhang T. (2023). Metabolomics Method in Understanding and Sensitizing Carbapenem-Resistant Acinetobacter baumannii to Meropenem. ACS Infectious Diseases, 10(1), 184-195.
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4

Quantification of Adenine Nucleotides in Trypanosoma Cells

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Control (transfected with scrambled sgRNA) and TcLetm1-KD epimastigotes were harvested and washed once with buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4 and 50 mM HEPES at pH 7.0). After washing, 1 × 108 cells per treatment were centrifuged and resuspended in 100 μl of buffer A, and then lysed on ice for 30 min by addition of 150 μl of 0.5 M HClO4. The lysates were neutralized (pH 6.5) by addition of 60 μl of 0.72 M KOH/0.6 M KHCO3. Samples were centrifuged at 1000 × g for 5 min and the supernatant was separated for adenine nucleotide determination. ATP, and ADP in extracted samples were quantified by a luciferin–luciferase bioluminescence assay in a plate reader using an ATP Determination Kit according to manufacturer’s instructions, with adenylate kinase and/or nucleoside-diphosphate kinase (NDK; Sigma). To determine the amount of adenine nucleotides, three measurements were taken of two different reactions for each sample by end point determination of the ATP concentration: one reaction without addition of any ATP-generating enzyme (for ATP), and a second reaction adding NDK (for ATP + ADP). The amount of ADP was obtained by subtracting the ATP from the (ATP + ADP) value.
dos Santos G.R., Leite A.C., Lander N., Chiurillo M.A., Vercesi A.E, & Docampo R. (2021). Trypanosoma cruzi Letm1 is involved in mitochondrial Ca2+ transport, and is essential for replication, differentiation, and host cell invasion. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(7), e21685.
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5

Mitochondrial Membrane Potential Assay

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Adenylate kinase, aminotriazole, Amplex Red, Ap5A, ATP, carbonylcyanide m-chlorophenylhydrazone (CCCP), cyclosporine A (CsA), EGTA, fatty acid-free BSA, glutamate, glucose-6-phosphate dehydrogenase, glycerol, hexokinase, malate, succinate, sucrose, mannitol, rotenone, NADP, phosphoenolpyruvate, pyruvate kinase, MgCl2, (NH4)2SO4, NaCl, Phenol red, and Tris were purchased from Sigma-Aldrich (USA). BAC (as a mixture of BACs with an average mol. weight of 360 and benzyldimethyldodecylammonium chloride as the prevalent species present) was from Fluka (Germany); Coomassie G-250 was from MP Biomedicals (USA); safranin O, KH2PO4, K2HPO4, KCl, and CaCl2 were from Merck (Germany); MitoTracker Green FM, and H2DCF-DA, and propidium iodide were from Life Technologies (USA). Other reagents of the highest quality available were from domestic suppliers. SkQ1 was a gift from Dr. G. A Korshunova.
Rogov A.G., Goleva T.N., Sukhanova E.I., Epremyan K.K., Trendeleva T.A., Ovchenkova A.P., Aliverdieva D.A, & Zvyagilskaya R.A. (2020). Mitochondrial Dysfunctions May Be One of the Major Causative Factors Underlying Detrimental Effects of Benzalkonium Chloride. Oxidative Medicine and Cellular Longevity, 2020, 8956504.
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