Kinase cell-free assay

AMPK can also be activated by a Ca2+-mediated pathway involving phosphorylation at Thr-172 by the Ca2+/calmodulin-dependent protein kinase, CaMKK 3. CaMKKa and CaMKK 3 were discovered as the upstream kinase for the calmodulin-dependent protein kinases-1 and -IV they both activate AMPK in a Ca2+/ calmodulin-dependent manner in cell-free assays, although CaMKK 3 appears to much more active against AMPK in intact cells. Expression of CaMKKa and CaMKK(3 primarily occurs in neural tissues, but CaMKKp is also expressed in some other cell types. Thus, the Ca2+-mediated pathway for AMPK activation has now been shown to occur in response to depolarization in rat neuronal tissue, in response to thrombin (acting via a Gq-coupled receptor) in endothelial cells, and in response to activation of the T cell receptor in T cells. 

In order to determine whether the released adenosine could serve as a precursor of human erythrocyte adenine nucleotides, a rabbit liver was labeled by perfusion with Hj -hypoxanthine as described. Washout perfusion with an isotonic balanced salt solution removed residual extracellular label. The labeled liver was then perfused by recirculating a 400ml washed human erythrocyte suspension for one hour. Erythrocytes were then collected and washed, and the liver was excised. Extracts were prepared and the purine nucleotides were assayed for distribution of radioactivity (Table II). Within the liver, the radioactivity was approximately evenly distributed between the adenine nucleotides and the hypoxanthine plus xanthine nucleotides, indicating again that extensive conversion of hypoxanthine to IMP to AMP can occur in the liver cell. Within the human erythrocyte, over 80 percent of the label appeared in the adenine nucleotides. Since IMP is not converted to AMP in that cell, the labeled adenosine formed in the liver from the perfused hypoxanthine must have been taken up by the human erythrocyte and converted to AMP by the adenosine kinase. Since free adenine, the only other possible precursor of human erythrocyte adenine nucleotides, was not detected in hypoxanthine perfused liver or in hepatic venous effluent from hypoxanthine perfused liver, a possible role is unlikely. 

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