Study on structure and function of myofibril-bound serine proteinase (MBP) using molecular cloning
| Project/Area Number |
13660203
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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 |
Fisheries chemistry
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| Research Institution | Nagasaki University |
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Principal Investigator |
HARA Kenji Nagasaki University, Fac. of Fish., Professor, 水産学部, 教授 (10039737)
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| Co-Investigator(Kenkyū-buntansha) |
OSATOMI Kiyoshi Nagasaki University, Fac. of Fish., Professor, 水産学部, 助教授 (40253702)
TACHIBANA Katsuyasu Nagasaki University, Fac. of Fish., Professor, 水産学部, 教授 (20171712)
ISHIHARA Tadashi Nagasaki University, Fac. of Fish., Professor, 水産学部, 教授 (40039722)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Myofibril-bound serine protease / Molecular cloning / Amino acid sequence / Carp / Lizard fish / Trypsin type protease / Modori / Serine proteinase inhibitor / mRNA発現 / 筋原繊維結合型セリンプロテアーゼ / N末端アミノ酸配列 / 火戻り / 精製 / プローブ |
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| Research Abstract |
Myofibril-bound serine proteinases (MBP) from carp and lizard fish were purified. Proteolysis of the two MBPs on myofibril proteins and their gel formation ability were investigated. MBPs readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBPs. The degradation effects of MBPs on actin, α-actinin and tropomyosin were studied by the imrnunoblotting method. Because of its myofibril-bound and myofibril protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.
A full-length cDNA clone encoding was amplified according to reverse transcription-polymerase chain reaction of RNA from ordinary muscle of carp. The sequence consisted of a 33 by 5'-non-coding region and a 726 by open reading frame which was followed by a 250 by 3'-non-coding region : The predicted protein consisted of 242 amino acids which was possibly processed to an active e
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nzyme of 222 amino acids that showed some similarity to other member of serine protease family. MBP contained the catalytic triad (His-60, Asp-106, and Ser-196) of trypsin-like proteases that has been characterized as the active site. Cys 167:181 (Met loop), and cys 192:216 (Ser loop), Compared to the amino acid compositions reported for trypsin-type serine proteases from all other sources, MBP molecule contained significant amounts of Lys (27 amino acid residues) per mole enzyme.
A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish. MBSPI was a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle. Less
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Report
(4 results)
Research Products
(6 results)
