| Summary: | 碩士 === 國立嘉義大學 === 生化科技學系研究所 === 101 === A wild range of cellular activities are regulated by protein phosphorylation/dephosphorylation catalyzed by two classes of enzymes, i.e. protein kinases and protein phosphatases. Although the extensive studies of protein kinases have been conducted and a rather deeper understanding of their molecular activities has been achieved, it seems not to be the same for protein phosphatases. For instance, the specific phosphorylation sites on certain target peptide sequences of many kinases have been deduced, but the consensus sequences for most phosphatases still remain unclear.
The current study is aimed to develop an experimental procedure that could isolate the total cellular substrates of the protein tyrosine phosphatase, LMW-PTP. In addition, the substrate specificity of two LMW-PTP isozymes, F and S forms, were also investigated.
For isolating phosphoproteins, we have tried three different column chromatographies, i.e. commercialized phosphoprotein purification kit, IMAC-Fe3+ column chromatography, and anion exchanger chromatography (High Q). The target phosphorylated proteins of LMW-PTP were identified and isolated by two strategies: (1) Protein extract was digested with active or inactive LMW-PTP, then identify the target proteins from the difference in the binding proteins. (2) in column digestion with LMW-PTP to elute out the target proteins. We have isolate 5 distinct protein targets. Two were F form targets, and the other three were S-form specific targets. For future study, it is believed that a much better result might be achieved if any of the following improvement could be obtained: (1) a better procedure for phosphorprotein enrichment; (2) a more sensitive and Mass compatible protein staining method; (3) a better way to increase the phosphorylation level in cellular proteins.
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