cDNA cloning of membrane-bound steroid receptor on oocyte maturation of fish
| Project/Area Number |
11640673
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
生物形態・構造
|
|---|
| Research Institution | Okazaki National Research Institutes |
|---|
Principal Investigator |
YSOHIKUNI Michiyasu Okazaki National Research Institutes, Associate Professor, 基礎生物学研究所, 助教授 (50210662)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
|
|---|
| Keywords | oocyte maturation / steroid hormone / hormone receptor / plasma membrane / metalloproteinase / egg membrane / fertilization / medaka / 細胞膜受容体 / 情報伝達 / キンギョ / 卵母細胞 |
|---|
| Research Abstract |
The aim of this research is the identification of the new membrane-bound steroid hormone receptor from fish ovarian tissues. The hormone binding activity of maturation-inducing steroid, 17α, 20β-dihydroxy-4-pregnen-3-one, was solubilized from membrane preparations of 8.4kg goldfish ovaries, and subjected to the purification process. A large scale 17α, 20β-DP-coupled Sepharose (100cm^3) column was newly prepared for gel retardation chromatography. The binding activity was purified by Q-Sepharaose FF anion exchange, S300HR gel filtration, POROS Q anion exchange, Phenyl Superose hydrophobic and 17α, 20β-DP get reterdtion chromatographies. In the final active fraction, fFive bands were detected by high sensitive fluorescent stain on SDS-PAGE.Apparent molecular sizes of these bands were estimated as 198, 110, 93, 67, 41KDa. Two major bands were further analyzed by in get digestion with trypsin and micro peptide seqencing. Two peptide fragments were determined amino acid sequences as follows ; IT (P) H (L) PDF from 93KDa band and E (T) V (D) EL (Y) AXLF from 67KDa band. However, these sequences were not interesting for the membrane steroid receptor molecule. A new metalloproteinase was found to induce Chorion-hardening at fertilization of fish egg. This protease was purified from egg cortical alveoli, detemined cDNA sequence and named Alveolin. Alveolin partially hydrolyzed large Chorion protein and induced the specific cross-linking with another chorion protein. The molecular structure of the first cross-linking was revealed as 1 : 1 linkage by two major Chorion proteins at the specific amino acid residues.
|
Report
(3 results)
Research Products
(4 results)
