It has been well-known that VILIP-1 but not VILIP-3 regulates the activity of
guanylyl cyclase-B. In order to identify the modulated region within VILIP-1
on regulating guanylyl cyclase-B activity, the recombinant myristoylated and
nonmyristoylated VILIPs (VILIP-1, VILIP-3, chimeric VILIPs, and mutant
VILIP-1) were prepared in the present study. The recombinant proteins were
purified using ion-exchanger chromatography followed by gel filtration. CD
spectra indicated that the secondary structure of VILIPs was dominant with
£\-helix, reflecting a well-conserved EF-hand structure. Tryptic digestion assay
and the fluorescence measurement showed that myristoylation, Ca2+ and Mg2+
differently induced the conformational changes of VILIPs. The results of gel
filtration chromatography reflected that the EF-3&4 of VILIP-1 and
myristoylation were involved in the dimerization of VILIP-1, and the dimer and
monomer were converted each other in a dynamic manner. The porcine brain
membrane binding assay and liposome binding assay showed that the binding
capability of VILIPs were markedly enhanced by myristoylation, Mg2+ and Ca2+.
Myristoylation and the intact EF-1 of VILIP-1 were found to essential for
the regulation of guanylyl cyclase activity in the presence of Mg2+ and Ca2+.
Taken together, theses results suggest that myristoylation and EF hand-1 of
VILIP-1 are the structural elements crucial for regulating the guanylyl cyclase
activity. In contrast to oligomerization of VILIP-1, Mg2+ and Ca2+ -induced
conformational changes of VILIP-1 and enhancement of the binding of VILIP-1
with membrane by Mg2+ and Ca2+ partly but not heavily involve in the action.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0718106-114037 |
| Date | 18 July 2006 |
| Creators | Wang, Li-Kuan |
| Contributors | none, none, Long-Sen Chang |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0718106-114037 |
| Rights | withheld, Copyright information available at source archive |
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