| Project/Area Number |
05640773
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
動物生理・代謝
|
|---|
| Research Institution | Tokyo Metropolitan University |
|---|
Principal Investigator |
IZUMI Susumu Tokyo Metropolitan University, Faculty of Science, Associate professor, 理学部, 助教授 (10145659)
|
|---|
| Project Period (FY) |
1993 – 1994
|
| Project Status |
Completed (Fiscal Year 1994)
|
| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
|
|---|
| Keywords | Insect / Silkworm / Bombyx / Chitin / Expression vector / Fusion protein / Cell adhension / Cuticle / N-アセチルグルコサアミン |
|---|
| Research Abstract |
A major larval cuitcle protein of Bombyx mori, termed "LCP30" is a basic protein with molecular weight 22k in mature form. The sequence analysis of the cloned LCP30 cDNA has shown the presence of an arginine-glycine-aspartic acid (RGD) tripeptide sequence, which is a consensus cell-adhesive motif occurring in many extracellular matrix proteins.
In the present work, native form of LCP30 was purified from the larval integument of B.mori and its molecular properties and physiological functions were studied.
1. LCP30 was purified to homogeneity from the guanidine-HCl extract of crude integuments of the fifth instar larvae through the steps of ammounium sulfate precipitation, ion exchange chromatography and preparative polyacrylamide gel elecctrophoresis.
2. Chitin-binding activity of LCP30 was qualitatively demonstrated by adsorption of LCP30 on chitin paper followed by immuno-detection of antigen by use of specific antibody against LCP30.
3. Chitin-binding activity of LCP30 was quantitatively determined by the assay method using the radiolabeled LCP30 and powdered chitin. Binding of LCP30 to chitin was little influenced by salt concentrations and divalent cations. Binding of the radiolabeled LCP30 to chitin was inhibited by the presence of N-acetvlchitobiose in the reaction mixture . Binding of the radiolabeled LCP30 to chitin was competed by the unlabeled LCP30.
4. The experiments employing the cultured cells of B.mori proved the cell attachment was demonstrated by the inhibition experiments using oligopeptide bearing RGD sequence as specific competitor. Cell attachment mediated by LCP30 required divalent cations.
5. Bacterially expressed beta-galactosidase/LCP30 fusion protein exhibited chitin-binding as well as cell-attachment activity, proiding promising results for future studies on the molecular mechanism of cuticle organization by use of artificially mutated LCPs.
|
