AMP Kinase

Kim, S.F., Huang, A.S., Snowman, A.M., Teuscher, C. and Snyder, S.H. (2007) From the coven Antipsychotic drug-induced weight gain mediated by histamine HI receptor-linked activation of hypothalamic AMP-kinase. Proceedings of the National Academy of Sciences of the United States of America, 104 (9), 3456-3459. 

Whereas CK rephosphorylates ADP using PCr as the phosphate donor, AK (also called myokinase or AMP kinase) uses another molecule of ADP as the phosphate donor. 

W. Zhu and E. J. Smart. Myristic Acid Stimulates Endothelial Nitric-oxide Synthase in a CD36- and an AMP Kinase-dependent Manner. J. Biol. Chem. 280 29543-29550 (2005). 

Arad, M., Benson, D. W., Perez-Atayde, A. R., McKenna, W. J., Sparks, E. A., Kanter, R. J., McGarry, K., Seidman, J. G. and Seidman, C. E., 2002, Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy, J Clin Invest, 109, pp 357-62. 

To apply this method to the problem at hand, the AMP kinase activity should be measured first. Clearly, it would be advantageous to carry out this assay under conditions similar to those that would be present when only ATP was added to the complex. Thus, one might set up a reaction mixture with about 1 mM ATP and with AMP in the nanomolar concentration range that is, at a concentration that would be expected if the AMP had been derived from reaction (2). In addition, to enable us to follow its fate, in reaction (3) the AMP should be added to the incubation mixture in a radiolabeled form. Reaction (3) is started by the addition of the enzyme complex, and samples should be removed from the incubation mixture at suitable intervals and injected onto the HPLC column for analysis. After separation, the eluent should be monitored by both radiochemical and UV (254 nm) detectors. 

If an AMP kinase is active, the following reactions would be expected to occur during the incubation ... 

Experiments have been carried out to demonstrate the method described above by means of a multienzyme complex. This complex was assayed first for the AMP kinase activity. A reaction mixture was prepared containing unlabeled ATP (1 mM) and radioactive [3H]AMP only. The reaction was started by the addition of the complex, and samples were removed and analyzed by HPLC. Chromatographic profiles, each representing the analysis of a sample removed from an incubation mixture at increasing times after the start of the incubation, are shown in Figure 10.4. Both optical density and radioactivity were determined. 

The profile of a sample taken from the incubation mixture early in the incubation shows two peaks, one of labeled AMP with a retention time of 2 minutes and the other unlabeled ATP with a retention time of 9 minutes. No products have been formed. The next sample, obtained after a 10 minute incubation, reveals the presence of small amounts of labeled ADP, indicating that the AMP kinase is active [see reaction (3)], since an ATPase would, of course, produce unlabeled ADP [see reaction (1)]. Samples taken after additional incubation reveal the continued loss of radiolabeled "AMP and an increase in radiolabeled ATP. These observations indicate that reaction (4) has been functioning and that the AMP kinase is now operating in the reverse direction, that is, according to reaction (5). 

With the presence of an AMP kinase in the complex established, it is necessary to determine whether an ATP pyrophosphohydrolase activity is present in the complex to catalyze reaction (2). For these experiments, a reaction mixture is prepared with unlabeled ATP only and the formation of AMP and ADP is followed. Since the myokinase is present, as well as any AMP formed from the pyrophosphohydrolase, the remaining ATP will be used by the myokinase, and ADP will be formed. Of course, this ADP might be formed directly by an ATPase according to reaction (1), and the AMP... 

Figure 10.11 AMP can be formed by adenosine kinase (1) in a reaction that uses ATP as the phosphate donor and forms ADP as the second reaction product. Alternatively, AMP can be deaminated to IMP by the enzyme AMP deaminase (2) and converted to inosine (INO) by a 5 -nucleotidase activity (3). Finally, AMP can be phosphorylated to ADP by the enzyme AMP kinase (4).

Figure 10.12 Comparison of the reaction products formed by AMP kinase with both AMP and its formycin analog formycin S -monophosphate as substrates.

ATP hydrolysis and this could be seen as beneficial competes with calcium and decreases contractility, inhibits nucleotidases and prevents further breakdown of AMP. AMP activates AMP kinase with subsequent increase in the rate of glycolysis and fatty acid oxidation. Figure 2. AMPK is responsible for the activation of glucose uptake and glycolysis during low-flow ischemia and seems to play an important protective role in limiting damage and apoptotic activity associated with ischemia and reperfusion in the heart.44... 

Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, et al. 2004. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428 569-574. 

NMR method in this case 15N-HSQC, depends on following the movement of cross-peaks as a small molecule is added. If a titration is performed, an NMR-XD can be extracted, as shown in the graph, (b) Ligand-detected STD NMR method (i) ID control spectrum of AMP/kinase (ii) STD spectrum of AMP/kinase only resonances of atoms that contact the protein are present in the STD spectrum (Hi) STD spectrum of ATP/kinase complex (iv) STD of ATP/kinase/competitor, the STD signal due to ATP is decreased because ATP is partially displaced from the binding site by the competitor, and new STD signals for the competitor appear, compared to spectrum (Hi)... 

Interestingly, (S) -FPMPA was far superior to (R) - FPMPA and (R) - PMPA far superior to (5) -PMPA in inhibiting MSV-induced cell-transformation and HIV replication, whereas for FPMPDAP, FPMPG and PMPG differences in activities of the two enantiomers were less apparent. This discrepancy in the adenine series of derivatives, notably of FPMPA, was attributed to the differential rates of phosphorylation of the two enantiomers by AMP kinase. 

Similar observations were made in a case-conholled study from Scotland [42] suggesting that the use of metformin may be associated with reduced risk of cancer (HR = 0.79). In Une with these observational studies are new experimental data [43] demonshating that metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. 

Zakikhani M, Dowling R, Fantus IG et al. Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer Res 2006 66 10269-10273. 

Figure 1. Purine salvage pathways of Leishmania species. Enzymes 1) phosphoribosyltransferase 2) adenine deaminase 3) guanine deaminase 4) adenosine deaminase 5) nucleoside kinase 6, nucleotidase 7) AMP deaminase 8) adenylosuccinate synthetase 9) adenylosuccinate lyase 10) AMP kinase 11) GMP kinase 12) IMP dehydrogenase 13) GMP synthetase 14) GMP reductase.

Adenosine Kinase AMP Kinase ADP Kinase Adenosine Deaminase Inosine Phosphorylase... 

AMP kinase of rabbit muscle was not inactivated by 1 mAf nominal initial levels of (I) (a 2-day-old sample) or of (II) (freshly prepared). 

Kramer, D.K., Al-Khalili, L., Guigas, B., et al. (2007) Role of AMP kinase and PPARdelta in the regulation of lipid and glucose metabolism in human skeletal muscle. Journal of Biological Chemistry, 282, 19313-19320. 

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