| Co-Investigator(Kenkyū-buntansha) |
DECKER Stuar ロックフュラー大学, 生化学部, 助教授
MINGーCHEH Li オクラホマ大学, 理学部, 准教授
NISHIYAMA Kazuo Miyazaki University Department of Biological Resource Science Assistant Professo, 農学部, 助手 (40164610)
SUIKO Masahito Miyazaki University Department of Biological Resource Science Associate Professo, 農学部, 助教授 (00128357)
LIU Ming-cheh The University of Oklahoma, U. S. A. Department of Chemistry and Biochemistry As
STUART Decker J. The Rockefeller University, U. S. A. Department of Biochemistry Assistant Profes
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| Research Abstract |
Post-translational protein modification by tyrosine sulfation has now been recognized to be widespread in animal cells and there is evidence indicating its involvement in modulation and modification of the biological activity of proteins, in influencing protein degradation, and in intracellular transport of proteins. Despite the considerable amount of effort made in recent years, the functional relevance of protein tyrosine sulfation, still, has not been fully elucidated. In an effort to verify the fate of the tyrosine sulfate from catabolized proteins in cells, the spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with[ ^<35>S]sulfate were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presense of tyramine-O-[ ^<35>S]Sulfate in addition to tyrosine-O-[ ^<35>S]sulfate in spent medium from human hepatoma cells. In contrast, only tyramine-O-[ ^<35>S]sulfate was observed in spent
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medium of 3Y1 rat fibroblasts. We could also recognize a 175 kDa membrane-bound protein in the bovine liver which exhibited binding affinity for tyrosine-O-sulfate (TyrS), suggesting a hypothesis of receptor mediated protein secretion using tyrosine sulfation for its signal, which was also supported by the fact that this membrane-bound TyrS-binding protein was found to be present in complex forms with several respective tyrosine-sulfated proteins, in freshly prepared bovine liver membrane lysate. Further we have purified and characterized a TyrS-binding protein (160 kDa) from bovine serum, which appears to be a good candidate as a putative carrier protein specific for tyrosine-sulfated proteins in the circulatory system, where the behavior of the tyrosine-sulfated proteins has not been elucidated. In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST) was characterized from a bovine liver Golgi preparation. Peptide substrates were synthesized and were sulfated with the TPST and Boc-Asp-Tyr-Val was found to be sulfated optimally. Less
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