| Project/Area Number |
01470149
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 | Kyushu University |
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Principal Investigator |
OHNO Motonori Kyushu University, Faculty of Science, Professor, 理学部, 教授 (30038434)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMOHIGASHI Yasuyuki Kyushu University, Faculty of Science, Res. Associate, 理学部, 助手 (00211293)
WAKI Michinori Kyushu University, Faculty of Science, Res. Associate, 理学部, 助手 (30037212)
AOYAGI Haruhiko Kyushu University, Faculty of Science, Assoc. Professor, 理学部, 助教授 (80037267)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1989: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Keywords | Trimeresurus flavoviridis / Phospholipase A_2 / Isozyme / Genetic engineering / cDNA / Genome DNA / ハブ毒 / アミノ酸配列 / アクロモバクタ-プロテア-ゼI / α→β転位 / イソ酵素 |
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| Research Abstract |
A highly reactive Asp-49-phospholipase A_2 (PLA_2) and two Lys-49-PLA_2, whose activities are only 1-2% that of Asp-49-PLA_2, from Trimeresurus flavoviridis venom contain 122 amino acid residues, and their sequences are 60% homologous and the positions of the 14 half-cystine residues are identical. Such PLA_2 isozymes provide an invaluable system for elucidating the structural elements required for eliciting catalytic function and various physiological functions. The aim of this research is to study the structure-function relationships of PLA_2 through synthesis of mutant proteins of the types of T. flavoviridis PLA_2. (1) As a step of this study, an active site His47-Asp99 couple in Asp-49-PLA_2 was investigated by pH-dependent methylation of His47 with methyl p-nitrobenzenesulfonate. Protonation of His47 appears to stabilize a transient state of catalysis. (2) Three cDNAs for Asp-49-PLA_2 and two Lys-49-PLA_2 were prepared. The amino acid sequence deduced from the uncleotide sequence
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of cDNA of Asp-49-PLA_2 was partially inconsistent with the amino acid sequence determined with the protein. The reinvestigation of the protein sequence established that the sequence from cDNA is correct. The nucleotide sequence of a cDNA encoding a PLA_2 which is similar to an Asp-49-PLA_2 called PL-X was also sequenced. Expression of the proteins using the cDNAs by recombinant DNA technology has not yet been successful. Intensive study is in progress. (3) Comparison of the four cDNAs encoding four different PLA_2 led to an interesting discovery that the nucleotide sequences of 5'- and 3'-noncoding regions are much more homologous than those of the coding regions. As a step for searching the roles of the noncoding regions in regulation and expression, analysis of genome DNA is on the way. One of nine clones obtained us under analysis. (4) Although expression of PLA_2 proteins has not yet been successful during the period, the finding of the unique nucleotide sequences for T. flavoviridis PLA_2 isozymes established the basis for future development. Less
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