1988 Fiscal Year Final Research Report Summary
Biochemical Studies on Human Inter- trypsin Inhibitor
| Project/Area Number |
62580106
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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 |
物質生物化学
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| Research Institution | Hokkaido University |
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Principal Investigator |
NAGASAWA Shigeharu Hokkaido University, Faculty of Pharmaceutical Sciences, 薬学部, 助教授 (70029958)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Kazuhiko Hokkaido University, Faculty of Pharmaceutical Sciences, 薬学部, 助手 (10113581)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Inter- trypsin Inhibitor / Plasma Protein / Protease / 阻害因子 |
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| Research Abstract |
Inter- trypsin inhibitor (ITI) is one of seven serine protease inhibitors present in human plasma. In the present study, we have established a practical method for isolation of ITI and attempted to determine polypeptide chain structure of ITI.
1) Purification of ITI. ITI was purified from human plasma by chromatographies with Q-Sepharose, DEAE-Sephacel, and heparin-Sepharose. By this method, about 50 mg of ITI were obtained from 1 L human plasma.
2) Polypeptide chain structure of ITI. Reduced as well as non-reduced ITI gave a single band on SDS-polyacrylamide gel electrophoresis of 210 kDa, suggesting ITI to be composed of a single polypeptide chain. However, the single chain ITI gave two N-terminal amino acid residues; Ser-Leu-Pro- and Ala-Val-Leu--. Some ITI preparations gave additional bands of 140 kDa and 70 kDa, which were separated by high performance liquid chromatography using a Mono-Q column. The 140 kDa fragment retains the trypsin inhibitory activity and its N-terminal was Ala-Val-Leu, while the N-terminal of 70 kDa fragment was Ser-Leu-Pro. These results suggested that ITI is composed of two chains of 140 and 70 kDa which are-connected with a unique linkage.
3) Chemical and enzymatic cleavage of ITI. Incubation of ITI at pH 12 for 60 min at 37 C resulted in the cleavage of ITI into two 70 kDa and 45 kDa fragments. Inhibitory activity was found to be associated with 45 kDa fragment. Treatment of the 45 kDa fragment with hyarulonidase resulted in the production of 25 kDa fragment having inhibitory activity. In addition, treatment of intact ITI with hyarulonidase produced 25 kDa inhibitory fragment and 170 kDa fragment. These results suggested that ITI is composed of three chains of 25,70,and 100 kDa and that the active domain of 25 kDa is connected to via carbohydrate chain of 20 kDa to the 100 kDa chain. Alkalinetreatment of ITI seems to cleave the linkages connecting the three polypeptide chains.
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