Structure and Function of the Phospholipase AィイD22ィエD2 Inhibitor Having Leucine-rich Repeats
| Project/Area Number |
10672074
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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 |
Biological pharmacy
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| Research Institution | Osaka University of Pharmaceutical Sciences |
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Principal Investigator |
INOUE Seiji Faculty of Pharmacy, Osaka University of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (70183184)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | Phospholipase AィイD22ィエD2 / Inhibitor / Venomous snake / Leucine-rich repeats / Tertiary structure / Serum / cDNA / Inhibition mechanism / 毒ヘビ |
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| Research Abstract |
1. A large-scale purification of phospholipase AィイD22ィエD2 inhibitors from the serum of Chinese mamushi and the crystallization of the inhibitors
We have purified three types of phospholipase AィイD22ィエD2 inhibitors (PLIα, PLIβ, and PLIγ) by the sequential chromatography on Blue-Sepharose FF, Q-Sepharose FF, Phenyl-Sepharose HP, and Superdex 200pg columns. Finally, 10 mg of the purified PLIα, 5 mg of the PLIβ, and 20 mg of the PLIγ could be obtained from 10 ml of the Chinese mamushi serum. We are determining the best crystallization conditions of these inhibitors by the standard technique of vapor diffusion with the hanging drop methods. By this time, we could not obtain the crystals suitable for X-ray crystallography.
2. Establishment of the bacterial expression system for PLAィイD22ィエD2 inhibitors and elucidation of their structure function relationships by means of the site-directed mutagenesis analysis
The PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Esc
… More
herichia coli. We could obtain the recombinant protein having the inhibition activity as strong as that of the natural PLIα. Alternatively, we have identified a PLIα-like protein (PLIα-LP) from the serum of the non-venomous striated snake and cloned its cDNA. The purified PLIα-LP was found to have no inhibitory activity toward any PLAィイD22sィエD2, although its amino acid sequence showed about 70% homology to that of Chinese mamushi PLIα. We have constructed the recombinant chimeric proteins between the PLIα-LP and Chinese mamushi PLIα and examined their inhibitory activity in order to specify the region responsible for the PLAィイD22ィエD2 inhibition. It was suggested that the CRD structure in the PLIα molecule played the basic structural roles such as the formation of trimeric structure and that the N-terminal and C-terminal regions of PLIα other than CRD were required for the interaction of PLIα to a PLAィイD22ィエD2 molecule.
3. Purification of leucine-rich αィイD22ィエD2 glycoprotein (LRG) from the human serum and the examination of its PLAィイD22ィエD2 inhibitory activity.
Using the synthesized peptide (LRG1-20) corresponding to the reported N-terminal 20 amino acid sequence of LRG as an antigen, we immunized a rabbit and obtained the anti-LRG1-20 antibody. The ELISA using this antibody was found to be a good tool for detecting LRG during the purification of LRG from human serum. Less
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Report
(3 results)
Research Products
(12 results)
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[Publications] Ohkura, N., Kitahara, Y., Inoue, S., Ikeda, K., and Hayahsi, K.: "Isolation and Amino Acid Sequence of a Phospholipase AィイD22ィエD2 inhibitor from the Blood Plasma of the Sea Krait, Laticauda semifasciata"J. Biochem.. 125 (2). 375-382 (1999)
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[Publications] Okumura, K., Masui, K., Inoue, S., Ikeda, K., and Hayashi, K.: "Purification, Characterization and cDNA Cloning of a Phospholipase AィイD22ィエD2 Inhibitor from the Serum of the Non-venomous Snake Elaphe quadrivirgata"Biochem. J.. 341 (1). 165-171 (1999)
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[Publications] Okumura, K., Inoue, S., Ohkura, N., Ikeda, K., and Hayashi, K.: "cDNA Cloning of the Two Subunits of Phospholipase AィイD22ィエD2 Inhibitor PLIγ from Blood Plasma of the Chinese Mamushi, Agkistrodon blomhoffii siniticus"IUBMB Life. 48 (1). 99-104 (1999)
Description
「研究成果報告書概要(欧文)」より
Related Report
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