1995 Fiscal Year Final Research Report Summary
Joint Study on Post-Translational Protein Tyrosine Sulfation
| Project/Area Number |
06044187
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Miyazaki University |
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Principal Investigator |
SUIKO Masahito Miyazaki University, Department of Biological Resource Science, Associate Professor, 農学部, 助教授 (00128357)
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| Co-Investigator(Kenkyū-buntansha) |
LIU Chau-ching The Rockefeller University, U.S.A., Department of Biochemistry, Assistant Profes, 生化学部, 助教授
LIU Ming-cheh The University of Texas, Health Center at Tyler, U.S.A., Department of Biochemis, ヘルスセンター, 準教授
ETO Nozomu Miyazaki University, Department of Biological Resource Science, Lecturer, 農学部, 講師 (90232959)
NISHIYAMA Kazuo Miyazaki University, Department of Biological Resource Science, Associate Profes, 農学部, 助教授 (40164610)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Tyrosine Sulfation / Posttranslational Modification / Sulfotransferase / Recombinant Protein / Hirudin / Golgi body / Microsome / Secreted Protein / Monoclonal Antibody |
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| Research Abstract |
The sulfation of protein is a post-translational modification by covalent attachment of sulfate. Among a number of possible funcitonal roles that have been proposed, the involvement of tyrosine sulfation in intracellular protein sorting and transport received strong support from our recent finding of Golgi located 175 k Da membrane-bound tyrosine-O-sulfate binding protein from bovine liver.Here we described the bindingproperties fo the tyrosine-O-sulfate (TyrS) receptor.
TyrS receptor was highly purified from bovine liver using a combination of TyrS-Affi-gel 10 affinity chromatography, hydroxylapatite chromatography and electroelution. The purified receptor exhibited an apparent molecular weight of 175k Da as determined by SDS-PAGE under reducing conditions. We established mouse monoclonal antibodies reactive to the purified receptor using P3-X63-Ag8-Ul cells as fusion partner cells. Two stable hybridoma clone secreting anti TyrS receptor monoclonal antibodies were obtained (R3-1, R2-4)
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We analyzed for the binding against TyrS,after solubilization the membrane-bound protein containing TyrS receptor. Trypsin was added to the suspension and the mixture was digested. To examine the ligand binding specificity, the reaction mixtures were incubated with TyrS-Affi-Gel 10.Then we analyzed by SDS-PAGE.It was apparent that three fragments of 65k, 63k and 59k Da have the ability to bind against TyrS.
TyrS receptor migrated as double protein bands with apparent molecular weights of ca. 175K upon SDS-PAGE.In order to obtain information concerning the sturctural form of these proteins and three fragments we investigated V8 peptide maps. Partial V8 protease mapping of these TyrS receptor were similar showing the homology. TyrS is a glycoprotein which has N-liked carbohydrate chain. So, TyrS receptor variously trimmed their carbohydrate chains by glycosidase showing the change of reactivity or loss of the ability to bind against TyrS.
It is concluded that TyrS receptor is a glycoprotein which is having a molecular weight of 175 K.The N linked carbohydrate residue of this protein is very important in recognition of the TyrS and has a functional contribution in receptor activity. Less
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