| AMP-activated protein kinase (AMPK), one serine/threonine kinase, has recently been discovered to be able to mediate abnormalities in glucose and fat metabolism and keep them in balance in body. AMPK plays a key role in regulation of carbohydrate and fat metabolism, functions as a 'fuel gauge' to monitor the alterations cellular energy status, and serves as a metabolic master switch. AMPK activation within cellular can result in following effects: stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake, inhibition of cholesterol synthesis, lipogenesis and triglyceride synthesis, regulation of insulin secretion by insulin secreting P cells, and enhancement of the insulin sensitive on insulin-acted target tissues. Therefore, AMPK signal pathway becomes a new attractive target for type II diabetes and obesity. Discovery of small molecular as activator of AMPK will be a potential method for therapy type II diabetes. Until recently, it has not been reported that HsAMPK-αl was expressed and charactered in the prokaryotic system. In this paper, an expression system of HsAMPK-α1(312) and HsAMPK-αl(FL) in the E.coli were established. And the phosphorylation characters of HsAMPK-α1(312) were studied in vitro. According to above mentioned results, we established two molecule-based high-throughput screening (HTS) assay for HsAMPK-α1(312) small molecular regulators on a basis of two respectively technologies: radioisotope and fluorescence polarization. Two inhibitors of HsAMPK-α1(312) were finally discovered from a compound library through a HTS by radioisotope assay. Those two inhibitors may be prospective lead compounds for inhibiting AMPK activity, which will provide a base for AMPK research on its action mechanism and functions in vivo.HsAMPK-α1(312) and HsAMPK-α1(FL) cDNA were obtained from an EST clone by PCR. They were cloned respectively into the pET42b and pGEX-KG expressionvector, which gave rise to the recombinant plasmids of pET42b-HsAMPK-α1(312) and pGEX-HsMPK-α1(FL) respectively. These recombinant plasmids were transformed into and expressed in the E.coli strain BL21(DE3) Codon Plus. GST-fusion recombinant proteins of GST-HsAMPK-α1 (312) and GST-HsAMPK-α1(FL) were soluble and purified separately through a glutathione affinity chromatography. GST were removed by thrombin cleavage. GST-HsAMPK-α1(FL) was discovered to be degraded, and the Western Blot results proved that the degradation took place at its C-terminal. HsAMPK-α1(FL) and the degraded protein were obtained through Q-sepharose chromatography. The results of N-terminal residue sequencing proved that HsAMPK-α1(FL) was degraded at its C-terminal. Furthermore, it was determined that the degradation site is A376-377R by Mass Spectrometry. The activity of this kinase was determined for SAMS phosphorylation using radioisotope assay. Study results showed that four proteins: GST-HsAMPK-α1(312), GST-HsAMPK-α1(FL) HsAMPK-1(312),HsAMPK-α1(376), had activity as making substrate SAMS phosphorylated, but HsAMPK-α1(FL) hadn't any activity. The Western-Blot results also proved that above proteins except HsAMPK-α1(FL) can make Thr172 phosphorylated respectively.The recombinant plasmid pMAL-CaMKKp was transformed and expressed in the E.coli BL21(DE3). The MBP-CaMKKp as a soluble fusion protein of Maltose Binding Protein(MBP) was purified through amylose resin affinity chromatography. MBP-CaMKKβ as a upstream kinase of AMPK, can make AMPK-α1 proteins phosphorylated, then phosphorylated AMPK can make its substrate SAMS phosphorylated. The phosphorylation of AMPK by CaMKKp reached saturation in 4 hours.The characteristics of HsAMPK-α1(312) were determined by radioisotope method.The apparent Km of SAMS and ATP was respectively 42 uM and 89 uM. The optimal concentration of Mg2+ was 5 mM; the optimal value of pH was 7.5, and the HsAMPK-α1(312) couldn't be activated by AMP. Based on these results, a molecule-based HTS assay was established using the radioisotope method according to the principle... |
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