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1

Annexin VI, a novel ATP-dependent protein. Alterations of phospholipid binding properties of annexin VI by nucleotides

100%
Danieluk M., Sikorski A. F., Pikula S., Bandorowicz-Pikula J.
Cellular and Molecular Biology Letters
|
1999
|
tom 04
|
nr 2
2

Annexin VI, a novel ATP-dependent, phospholipid-binding protein

100%
Danieluk M., Sikorski A. F., Pikula S., Bandorowicz-Pikula J.
Cellular and Molecular Biology Letters
|
1999
|
tom 04
|
nr 4
The determination of surface pressure (π) of a phosphatidylserine (PS) monolayer is used to study the interactions between specific phospholipid classes and various proteins. In the present study we show that ATP, but not ADP, in milimolar concentration ranges stimulate the increase of Δπ in a PS monolayer evoked by annexin VI (AnxVI)/Ca2+ at a moderate initial π (~11 mN/m). The obtained results are consistent with ATP being a functional ligand for AnxVI. To further study the ATP binding site of AnxVI, we have used fluorescein 5’-isothiocyanate (FITC). This is useful in the characterization of nucleotide-binding sites of many membrane integral and cytosolic proteins. Under our experimental conditions FITC did not affect the binding of AnxVI to membranes but abolished the interaction of the protein with ATP insolubilized on agarose. This observation can be interpreted in terms of AnxVI possessing an ATP-binding site functionally similar to nucleotide-binding domains characterized in other ATP-dependent proteins. We also provide evidence that two AnxVI isoforms are expressed constitutively in porcine liver differ from each other in respect to their ATP binding properties.
3

Annexin VI: an intracellular target for ATP

100%
Bandorowicz-Pikula J., Danieluk M., Wrzosek A., Bus R., Buchet R., Pikula S.
Acta Biochimica Polonica
|
1999
|
tom 46
|
nr 3
Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+ -dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.
4

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

100%
Danieluk M., Bus R., Pikula S., Bandorowicz-Pikula J.
Acta Biochimica Polonica
|
1999
|
tom 46
|
nr 2
Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to bind ATP in vitro with a Kd in the low micromolar concentration range. However, this protein does not contain within its primary structure any ATP-binding consensus motifs found in other nucleotide-binding proteins. In addition, binding of ATP to AnxVI resulted in modulation of AnxVI function, which was accompanied by changes in AnxVI affinity to Ca2+ in the presence of ATP. Using limited proteolytic digestion, purification of protein fragments by affinity chromatography on ATP-agarose, and direct sequencing, the ATP-binding site of AnxVI was located in a C-terminal half of the AnxVI molecule. To further study AnxVI-nucleotide interaction we have employed a functional nucleotide analog, Cibacron blue 3GA (CB3GA), a triazine dye which is commonly used to purify multiple ATP-binding proteins and has been described to modulate their activities. We have observed that AnxVI binds to CB3GA immobilized on agarose in a Ca2+-dependent manner. Binding is reversed by EGTA and by ATP and, to a lower extent, by other adenine nucleotides. CB3GA binds to AnxVI also in solution, evoking reversible aggregation of protein molecules, which resembles self-association of AnxVI molecules either in solution or on a membrane surface. Our observations support earlier findings that AnxVI is an ATP-binding protein.
5

Interaction of annexin VI with membranes. Regulation by ATP in vitro

84%
Bandorowicz-Pikula J., Danieluk M., Wrzosek A., Bus R., Pikula S.
Cellular and Molecular Biology Letters
|
1998
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tom 03
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nr 4
Porcine liver annexin VI (AnxVI) has recently been described to bind in vitro ATP. The binding of nucleotide to protein is accompanied by modulation of AnxVI function, such as its' interaction with F-actin and membranes. In the present report, we show that ATP modulates AnxVI-driven aggregation of phosphatidylserine (PS) liposomes. In addition, we provide evidence using circular dichroism (CD) that the interaction of AnxVI with ATP evokes changes in secondary structure of the protein. The functional implications of these changes are also discussed.
6

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

84%
Kirilenko A., Golczak M., Pikula S., Bandorowicz-Pikula J.
Acta Biochimica Polonica
|
2001
|
tom 48
|
nr 4
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')-0-(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 uM 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.
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