1996 Fiscal Year Final Research Report Summary
On searching the target proteins for mastoparan from bovine brain
| Project/Area Number |
06680776
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Tokyo Metropolitan College of Allied Medical Sciences |
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Principal Investigator |
KASAI Hisataka Tokyo Metropolitan College of Allied Medical Sciences, Division of Liberal Arts/Professor, 一般教養科, 教授 (80087163)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Hisashi Aoyama Gakuin University, Department of Chemistry/Professor, 理工学部・化学科, 教授 (70082815)
SUDA Haruhiko Tokyo Metropolitan College of Allied Medical Sciences, Division of Occupational, 作業療法学科, 教授 (40051784)
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| Project Period (FY) |
1994 – 1996
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| Keywords | Mastoparan / Exocytosis / Affinity Chromatography / GTPase / HPLC / Solid-phase Synthesis / GTPアーゼ |
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| Research Abstract |
In this study, we planned mastoparan analogue(LLL-mastoparan-G) as an affinity ligand in which the N-terminal region of mastoparan is modified and the C-terminal region is extended by Gly. The affinity resin with the ligand was prepared by stepwise elongation method using the peptide synthesizer, in a manner that it is coupled to amino resin sequentially from the C-terminal to the N-terminal amino acid. The structure of the affinity ligand obtained after cleavage was analyzed by use of peptide sequencer and high-resolution positive FAB-MS spectrometer. The data for peptide sequence and elemental composition corresponded closely to theoretical ones.
It was shown by affinity chromatography and SDS-PAGE that calmodulin bound to the affinity ligand in the presence of calcium ion. Furthermore, HPLC-type affinity chromatography was devised in addition to the open column type.
We searched for the target proteins for mastoparan from bovine brain by newly prepared affinity resin. Our affinity chromatography system showed that unique proteins bound to the affinity-ligand only in the presence of calcium ion. Its molecular weight was estimated as about 26k Da. The detected proteins bands were eluted from gels by use of Max-yield electroeluter, or transferred to PVDF membrane by western blotting. Amino acid analysis and protein sequence analysis of the proteins eluted in the manner described above was also attempted.
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