| Summary: | 碩士 === 國立交通大學 === 生物科技學系 === 107 === Protein tyrosine sulfation (PTS) is a common post-translational modification. 875 among 4256 proteins have been identified to undergo the PTS on their tyrosine residues. Tyrosylprotein sulfotransferase (TPST; Enzyme Commission number: 2.8.2.20) is the type II trans-Golgi membrane enzyme responsible for the PTS, delineating the transfer of the sulfonate group (SO3)- to specific tyrosine residues in target peptides/proteins to form O-sulfation compound. The sulfation activity plays essential roles in pathogen infection, hemostasis and protein–protein interactions. In order to understand the importance of PTS and tyrosine-sulfated proteins in the life biology, the preparation and characterization of TPST is crucial. In this thesis, Escherichia coli BL21(DE3) was used to produce the recombinant of rat phenol sulfotransferase (PST) and Drosophila melanogaster TPST (TPST), and they were purified to more than 95% homogeneity. The TPST sulfation on small P-selectin glycoprotein ligand-1 peptide (PSGL-1, ATEYEYLDYDFL) affected by five divalent metal ions: calcium (Ca2+), cobalt (Co2+), magnesium (Mg2+), manganese (Mn2+) and zinc (Zn2+) at pH 6.5 and 37oC was determined by phenol sulfotransferase–tyrosylprotein sulfotransferase (PST-TPST) coupled enzyme system through two assays. The former was enzyme-linked immunosorbent assay (ELISA)-based detection on the preparation of sulfated protein at A450 nm. The latter was based on continuously increasing fluorescence of 4-methylumbilliferone (MU) signal. Typically, one liter of Luria−Bertani (LB) broth purified through the His-Trap affinity column with only one chromatography step produced 14.2 mg TPST, its specific activity: 0.350 A450/min/mg or 7.87 nmole/min/mg. Besides, all of the metal cations enhanced the binding ability of PSGL-1 towards TPST, their Km values lower than apo-TPST. Only Zn2+ strongly inhibited the TPST activity, the IC50: 40-50 µM, whereas the other metals enhanced the sulfation activity. The catalytic efficiency of TPST was risen up to 3-fold with the assistance of Ca2+ and Co2+. Together with the available structures of human TPST, this research proposed specific linking interactions between metal-enhancer or metal-inhibitor with amino-acid residues of TPST. This thesis is facilitating the deeper understanding of TPST, which expands more aspects on the PTS regulation.
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