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GTP-binding properties of the membrane-bound form of porcine liver annexin VI.
- Source :
-
Acta biochimica Polonica [Acta Biochim Pol] 2001; Vol. 48 (4), pp. 851-65. - Publication Year :
- 2001
-
Abstract
- Annexin VI (AnxVI) of molecular mass 68-70 kDa belongs to a multigenic family of ubiquitous Ca2+- and phospholipid-binding proteins. In this report, we describe the GTP-binding properties of porcine liver AnxVI, determined with a fluorescent GTP analogue, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP). The optimal binding of TNP-GTP to AnxVI was observed in the presence of Ca2+ and asolectin liposomes, as evidenced by a 5.5-fold increase of TNP-GTP fluorescence and a concomitant blue shift (by 17 nm) of its maximal emission wavelength. Titration of AnxVI with TNP-GTP resulted in the determination of the dissociation constant (Kd) and binding stoichiometry that amounted to 1.3 microM and 1:1 TNP-GTP/AnxVI, mole/mole, respectively. In addition, the intrinsic fluorescence of the membrane-bound form of AnxVI was quenched by TNP-GTP and this was accompanied by fluorescence resonance energy transfer (FRET) from AnxVI Trp residues to TNP-GTP. This indicates that the GTP-binding site within the AnxVI molecule is probably located in the vicinity of a Trp-containing domain of the protein. By controlled proteolysis of human recombinant AnxVI, followed by purification of the proteolytic fragments by affinity chromatography on GTP-agarose, we isolated a 35 kDa fragment corresponding to the N-terminal half of AnxVI containing Trp192. On the basis of these results, we suggest that AnxVI is a GTP-binding protein and the binding of the nucleotide may have a regulatory impact on the interaction of annexin with membranes, e.g. formation of ion channels by the protein.
- Subjects :
- Animals
Calcium metabolism
Circular Dichroism
Dose-Response Relationship, Drug
Escherichia coli metabolism
Guanosine Triphosphate pharmacology
Humans
Kinetics
Protein Binding
Protein Conformation
Protein Isoforms
Protein Structure, Tertiary
Recombinant Proteins chemistry
Spectrometry, Fluorescence
Swine
Tryptophan chemistry
Annexin A6 chemistry
Annexin A6 metabolism
Guanosine Triphosphate analogs & derivatives
Guanosine Triphosphate metabolism
Liver metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0001-527X
- Volume :
- 48
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Acta biochimica Polonica
- Publication Type :
- Academic Journal
- Accession number :
- 11995996