Preferences help
enabled [disable] Abstract
Number of results
2001 | 48 | 4 | 851-865
Article title

GTP-binding properties of the membrane-bound form of porcine liver annexin VI.

Title variants
Languages of publication
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 μM 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.

Physical description
  • Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, L. Pasteura 3, 02-093 Warszawa, Poland
  • Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, L. Pasteura 3, 02-093 Warszawa, Poland
  • Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, L. Pasteura 3, 02-093 Warszawa, Poland
  • Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, L. Pasteura 3, 02-093 Warszawa, Poland
  • Abdulla, Y.H. (2001) A plausible function of the prion protein: Conjectures and a hypothesis. Bioessays 23, 456-462.
  • Avila-Sakar, A.J., Creutz, C.E. & Kretsinger, R.H. (1998) Crystal structure of bovine annexin VI in a calcium-bound state 1. Biochim. Biophys. Acta 1387, 103-116.
  • Avila-Sakar, A.J., Kretsinger, R.H. & Creutz, C.E. (2000) Membrane-bound 3D structures reveal the intrinsic flexibility of annexin VI. J. Struct. Biol. 130, 54-62.
  • Ayala-Sanmartin, J. (2001) Cholesterol enhances phospholipid binding and aggregation of annexins by their core domain. Biochem. Biophys. Res. Commun. 283, 72-79.
  • Babiychuk, E.B. & Draeger, A. (2000) Annexins in cell membrane dynamics. Ca2+ -regulated association of lipid microdomains. J. Cell Biol. 150, 1113-1124.
  • Babiychuk, E.B., Palstra, R.J., Schaller, J., Kampfer, U. & Draeger, A. (1999) Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells. J. Biol. Chem. 274, 35191-35195.
  • Bandorowicz, J., Pikuła, S. & Sobota, A. (1992) Annexins IV (p32) and VI (p68) interact with erythrocyte membranes in a calcium-dependent manner. Biochim. Biophys. Acta 1105, 201-206.
  • Bandorowicz-Pikuła, J. (1998) A nucleotide-binding domain of porcine liver annexin VI. Proteolysis of annexin VI labeled with 8-azido-ATP, purification of proteolytic fragments by affinity chromatography on ATP-agarose and fluorescence studies. Mol. Cell. Biochem. 181, 11-20.
  • Bandorowicz-Pikuła, J. & Awasthi, Y.C. (1997) Interaction of annexins IV and VI with ATP. An alternative mechanism by which a cellular function of these calcium- and membrane-binding proteins is regulated. FEBS Lett. 409, 300-306.
  • Bandorowicz-Pikuła, J., Wrzosek, A., Pikuła, S. & Awasthi, Y.C. (1997a) Fluorescence spectroscopic studies on interactions between porcine liver annexin VI and nucleotides - a possible role for a tryptophan residue. Eur. J. Biochem. 248, 238-244.
  • Bandorowicz-Pikuła, J., Wrzosek, A., Makowski, P. & Pikuła, S. (1997b) The relationship between the binding of ATP and calcium to annexin IV. Effect of nucleotide on the calcium-dependent interaction of annexin with phosphatidylserine. Mol. Membr. Biol. 14, 179-186.
  • Bandorowicz-Pikuła, J. & Pikuła, S. (1998a) Annexins and ATP in membrane traffic: A comparison with membrane fusion machinery. Acta Biochim. Polon. 45, 721-733.
  • Bandorowicz-Pikuła, J. & Pikuła, S. (1998b) Modulation of annexin VI-driven liposome aggregation by ATP. Biochimie (Paris) 80, 613-620.
  • Bandorowicz-Pikuła, J. & Pikuła, S. (1998c) Adenosine 5'-triphosphate - a new regulator of annexin VI function. Acta Biochim. Polon. 45, 735-744.
  • Bandorowicz-Pikuła, J., Wrzosek, A., Danieluk, M., Pikuła, S. & Buchet, R. (1999a) Changes in secondary structure of annexin VI upon ATP binding, as investigated by photochemical release of nucleotide, infrared difference, circular dichroism and fluorescence spectroscopies. Biochem. Biophys. Res. Commun. 263, 775-779.
  • Bandorowicz-Pikuła, J., Danieluk, M., Wrzosek, A., Buś, R., Buchet, R. & Pikuła, S. (1999b) Annexin VI: An intracellular target for ATP. Acta Biochim. Polon. 46, 801-812.
  • Bandorowicz-Pikuła, J., Buchet, R. & Pikuła, S. (2001) Annexins as nucleotide-binding proteins: Facts and speculations. BioEssays 23, 170-178.
  • Baubichon-Cortay, H., Baggetto, L.G., Dayan, G. & Di Pietro, A. (1994) Overexpression and purification of the carboxyl-terminal nucleotide-binding domain from mouse P-glycoprotein. Strategic location of a tryptophan residue. J. Biol. Chem. 269, 22983-22989.
  • Benevolensky, D., Belikova, Y., Mohammadzadeh, R., Trouve, P., Marotte, F., Russo-Marie, F., Samuel, J.L. & Charlemagne, D. (2000) Expression and localization of the annexins II, V, and VI in myocardiumfrom patients with end-stage heart failure. Lab. Invest. 80, 123-133.
  • Benz, J., Bergner, A., Hofmann, A., Demange, P., Gottig, P., Liemann, S., Huber, R. & Voges, D. (1996) The structure of recombinant human annexin VI in crystals and membrane-bound. J. Mol. Biol. 260, 638-643.
  • Bianchi, R., Giambanco, I., Ceccarelli, P., Pula, G. & Donato, R. (1992) Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins. FEBS Lett. 296, 158-162.
  • Bradford, M.M. (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
  • Caohuy, H., Srivastava, M. & Pollard, H.B. (1996) Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion. Proc. Natl. Acad. Sci. U.S.A. 93, 10797-10802.
  • Cartailler, J.P., Haigler, H.T. & Luecke, H. (2000) Annexin XII E105K crystal structure: Identification of pH-dependent switch for mutant hexamerization. Biochemistry 39, 2475-2483.
  • Cheng, K. & Koland, J.G. (1996) Nucleotide-binding by the epidermal growth factor receptor protein-tyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe. J. Biol. Chem. 271, 311-318.
  • Chow, A., Davis, A.J. & Gawler, D.J. (2000) Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120GAP C2 domain. FEBS Lett. 469, 88-92.
  • Cohen, B.E., Lee, G., Arispe, N. & Pollard, H.B. (1995) Cyclic 3'-5'-adenosine monophosphate binds to annexin I and regulates calcium-dependent membrane aggregation and ion channel activity. FEBS Lett. 377, 444-450.
  • Cuervo, A.M., Gomes, A.V., Barnes, J.A. & Dice, J.F. (2000) Selective degradation of annexins by haperone-mediated autophagy. J. Biol. Chem. 275, 33329-33335.
  • Danieluk, M., Pikuła, S. & Bandorowicz-Pikuła, J. (1999a) Annexin VI interacts with adenine nucleotides and their analogs. Biochimie (Paris) 81, 717-726.
  • Danieluk, M., Buś, R., Pikuła, S. & Bandorowicz-Pikuła, J. (1999b) Affinity labeling of annexin VI with a triazine dye, Cibacron blue 3GA. Probable interaction of the dye with C-terminal nucleotide-binding site within the annexin molecule. Acta Biochim. Polon. 46, 419-429.
  • Danieluk, M., Golczak, M., Pikuła, S. & Bandorowicz-Pikuła, J. (2001) UDP hydrolase activity associated with the porcine liver annexin fraction. Biochim. Biophys. Acta 1526, 70-76.
  • Davis, A.J., Butt, J.T., Walker, J.H., Moss, S.E. & Gawler, D.J. (1996) The Ca2+ dependent lipid binding domain of P120GAP mediates protein-protein interactions with Ca2+ dependent membrane-binding proteins. Evidence for a direct interaction between annexin VI and P120GAP. J. Biol. Chem. 271, 24333-24336.
  • Diaz-Munoz, M., Hamilton, S.L., Kaetzel, M.A., Hazarika, P. & Dedman, J.R. (1990) Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin). J. Biol. Chem. 265, 15894-15899.
  • Dice, J.F. (1990) Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem. Sci. 15, 305-309.
  • Fleet, A., Ashworth, R., Kubista, H., Edwards, H., Bolsover, S., Mobbs, P. & Moss, S.E. (1999) Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific. Biochem. Biophys. Res. Commun. 260, 540-546.
  • Giambanco, I., Verzini, M. & Donato, R. (1993) Annexins V and VI in rat tissues during post-natal development: Immunochemical measurements. Biochem. Biophys. Res. Commun. 196, 1221-1226.
  • Golczak, M., Kicińska, A., Bandorowicz-Pikuła, J., Buchet, R., Szewczyk, A. & Pikuła, S. (2001a) Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules. FASEB J. 15, 1083-1085.
  • Golczak, M., Kirilenko, A., Bandorowicz-Pikuła, J. & Pikuła, S. (2001b) Conformational states of annexin VI in solution induced by acidic pH. FEBS Lett. 496, 49-54.
  • Golczak, M., Kirilenko, A., Bandorowicz-Pikuła, J. & Pikuła, S. (2001c) N- and C-terminal halves of human annexin VI differ in ability to form low pH-induced ion channels. Biochem. Biophys. Res. Commun. 284, 785-791.
  • Gonzalo, P., Sontag, B., Lavergne, J.P., Jault, J.M. & Reboud, J.P. (2000) Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides. Biochemistry 39, 13558-13564.
  • Hawkins, T.E., Merrifield, C.J. & Moss, S.E. (2000) Calcium signaling and annexins. Cell Biochem. Biophys. 33, 275-296.
  • Huang, S.G. & Klingenberg, M. (1995) Fluorescent nucleotide derivatives as specific probes for the uncoupling protein: Thermodynamics and kinetics of binding and the control by pH. Biochemistry 34, 349-360.
  • Huang, S.G., Weisshart, K. & Fanning, E. (1998) Characterization of the nucleotide binding properties of SV40 T antigen using fluorescent 3'(2')O (2,4,6-trinitrophenyl)adenine nucleotide analogues. Biochemistry 37, 15336-15344.
  • Isas, J.M., Cartailler, J.P., Sokolov, Y., Patel, D.R., Langen, R., Luecke, H., Hall, J.E. & Haigler, H.T. (2000) Annexins V and XII insert into bilayers at mildly acidic pH and form ion channels. Biochemistry 39, 3015-3022.
  • Ishitsuka, R., Kojima, K., Utsumi, H., Ogawa, H. & Matsumoto, I. (1998) Glycosaminoglycan binding properties of annexin IV, V, and VI. J. Biol. Chem. 273, 9935-9941.
  • Jones, P.G., Moore, G.J. & Waisman, D.M. (1992) A nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling. J. Biol. Chem. 267, 13993-13997.
  • Kaetzel, M.A., Pula, G., Campos, B., Uhrin, P., Horseman, N. & Dedman, J.R. (1994) Annexin VI isoforms are differentially expressed in mammalian tissues. Biochim. Biophys. Acta 1223, 368-374.
  • Kirilenko, A., Golczak, M., Pikuła, S., Buchet, R. & Bandorowicz-Pikuła, J. (2002) GTP-induced membrane binding and ion channel activity of annexin VI. Is annexin VI a GTP biosensor? Biophys. J. 82, 2737-45.
  • Kourie, J.I. & Wood, H.B. (2000) Biophysical and molecular properties of annexin-formed channels. Prog. Biophys. Mol. Biol. 73, 91-134.
  • Köhler, G., Hering, U., Zschörnig, O. & Arnold, K. (1997) Annexin V interaction with phosphatidylserine-containing vesicles at low and neutral pH. Biochemistry 36, 8189-8194.
  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  • Lakovicz, J.R. (1983) Principles of Fluorescence Spectroscopy. Plenum, New York
  • Lavialle, F., Rainteau, D., Massey-Harroche, D. & Metz, F. (2000) Establishment of plasma membrane polarity in mammary epithelial cells correlates with changes in prolactin trafficking and in annexin VI recruitment to membranes Biochim. Biophys. Acta 1464, 83-94.
  • Lesieur, C., Frutiger, S., Hughes, G., Kellner, R., Pattus, F. & van der Goot, F.G. (1999) Increased stability upon heptamerization of the pore-forming toxin aerolysin. J. Biol. Chem. 274, 36722-36728.
  • Luckcuck, T., Trotter, P.J. & Walker, J.H. (1998) Localization of annexin VI in the adult and neonatal heart. Cell Biol. Int. 22, 199-205.
  • Moczydlowski, E.G. & Fortes, P.A.G. (1981) Characterization of 2',3'-O- (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP side of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP. J. Biol. Chem. 256, 2346-2356.
  • Morgan, R.O., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., Balsara, B.R., Testa, J.R. & Fernandez, M.P. (1999) Novel human and mouse annexin A10 are linked to the genome duplications during early chordate evolution. Genomics 60, 40-49.
  • Morrisey, J.H. (1981) Silver staining for protein in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117, 307-310.
  • Moss, S.E. & Crumpton, M.J. (1990) Alternative splicing gives rise to two forms of the p68 Ca2+ binding protein. FEBS Lett. 261, 299-302.
  • Orito, A., Kumanogoh, H., Yasaka, K., Sokawa, J., Hidaka, H., Sokawa, Y. & Maekawa, S. (2001) Calcium-dependent association of annexin VI, protein kinase C, and neurocalcin on the raft fraction derived from the synaptic plasma membrane of rat brain. J. Neurosci. Res. 64, 235-241.
  • Pollard, H.B., Caohuy, H., Minton, A.P. & Srivastava, M. (1998) Synexin (annexin VII) hypothesis for Ca2+/GTP-regulated exocytosis. Adv. Pharmacol. 42, 81-87.
  • Prevost, G., Mourey, L., Colin, D.A. & Menestrina, G. (2001) Staphylococcal pore-forming toxins. Curr. Top. Microbiol. Immunol. 257, 53-83.
  • Provencher, S.W. & Glockner, J. (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20, 33-37.
  • Prusiner, S.B. (2001) Shattuck lecture - neurodegenerative diseases and prions. N. Engl. J. Med. 344, 1516-1526.
  • Randak, C., Neth, P., Auerswald, E.A., Assfalg-Machleidt, I., Rosher, A.A., Hadorn, H.B. & Machleidt, W. (1996) A recombinant polypeptide model of the second predicted nucleotide-binding fold of thecystic fibrosis transmembrane conductance regulator is a GTP-binding protein. FEBS Lett. 398, 97-100.
  • Russo-Marie, F. (1999) Annexin V and phospholipid metabolism. Clin. Chem. Lab. Med. 37, 287-291.
  • Rand, J.H. (2000) The annexinopathies: A new category of diseases. Biochim. Biophys. Acta 1498, 169-173.
  • Schoenmakers, T.J.M., Visser, G.J., Flik, G. & Theuvenet, A.P.R. (1992) CHELATOR: An improved method for computing metal ion concentrations in physiological solutions. BioTechniques 12, 870-874.
  • Smith, P.D., Davies, A.A., Crumpton, M.J. & Moss, S.E. (1994) Structure of the human annexin VI gene. Proc. Natl. Acad. Sci. U.S.A. 91, 2713-2717.
  • Sopkova-De Oliveira Santos, J., Fischer, S., Guilbert, C., Lewit-Bentley, A. & Smith, J.C. (2000) Pathway for large-scale conformational change in annexin V. Biochemistry 39, 14065-14074.
  • Tagoe, C.E., Boustead, C.M., Higgins, S.J. & Walker, J.H. (1994) Characterization and immunolocalization of rat liver annexin VI. Biochim. Biophys. Acta 1192, 272-280.
  • Thomas, P.J., Shenbagamurthi, P., Ysern, X. & Pedersen, P.L. (1991) Cystic fibrosis transmembrane conductance regulator: Nucleotide binding to a synthetic peptide. Science 251, 555-557.
  • Thorin, B., Gache, G., Dubois, T., Grataroli, R., Domingo, N., Russo-Marie, F. & Lafont, H. (1995) Annexin VI is secreted in human bile. Biochem. Biophys. Res. Commun. 209, 1039-1045.
  • Towbin, H., Staehelin, T. & Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354.
  • Turpin, E., Russo-Marie, F., Dubois, T., de Paillerets, C., Alfsen, A. & Bomsel, M. (1998) In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner. Biochim. Biophys. Acta 1402, 115-130.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.