1990 Fiscal Year Final Research Report Summary
Studies on Post-Translational Tyrosine Sulfation
| Project/Area Number |
01560102
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | Miyazaki University |
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Principal Investigator |
SUIKO Masahito Department of Biological Resource Science Associate Professor, 農学部, 助教授 (00128357)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Tyrosine sulfation / Post-translational modification / Golgi body / Bovine liver / Microsome / Cholecystokinin / Sulfotransferase / Sulfatase |
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| Research Abstract |
Sulfation in animal cells plays an important role in the metabolism of many exogenous and endogenous compounds such as phenol, catechol, steroid derivatives etc. And in post-translational modification of proteins on the tyrosine residue and on carbohydrate moieties. These reactions are catalyzed by distinct enzymes viz. Sulfotransferases, that transfer the sulfate moiety of 3'-Phospho-Adenosine-5'-Phosphosulfate (PAPS) to a hydroxyl group of an acceptor substrate. Various sulfotransferases have been demonstrated to be located in both the cytosol and membrane organelles of cells. However, it is now clear that post-translation tyrosine sulfation is catalyzed in most cases by membrane bound enzymes which are mostly located in the Golgi. This research was undertaken to study the Golgi located tyrosylprotein sulfotransferase in bovine liver and to develop an efficient system for tyrosine sulfation of recombinant proteins.
Upon investigating the bovine liver microsomes we could identify a nov
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el membrane bound Phenol Sulfotransferase (PST) catalyzing sulfation of simple phenol derivatives, dopamine and tyramine, and showing a poor thermostability and what could be categorized as a "monoamine" form of phenol sulfotransferase.
TPST has been characterized from the bovine liver Golgi membranes with activity on Z-Glu-Tyr, similar di and tri peptides and Boc-CCK-8. Among the substrates tested, peptides having a Asp on the N side of Tyr showed a higher reactivity than the peptides having a Glu at this position. Sulfation could not be detected on a peptide with a lysyl residue at the said position. Therefore it was postulated that substrate recognition of TPST, does depend on the amino acid sequence around the tyrosine residue.
Hirudin, though has two Tyr residues is sulfated onl on the Tyr at position 63. However when hirudin is expressed in yeast, sulfation could not be observed on tyrosine 63 residue. Hence it is necessary to convert this recombinant product by in-vitro sulfation to its natural form. Sulfation of hirudin on the tyrosine 63 has been achieved with a heterologous TPST (bovine liver) with no sulfation on the tyrosine 3 residue. This confirmed that bovine liver TPST could selectively sulfate, tyrosine residues on specific amino acid sequences, in recombinant proteins. Less
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