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J Bandorowicz-Pikuła

Publications and source records attributed to J Bandorowicz-Pikuła.

At least 19 recordsLinked to original sources
PubMed2001

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

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.

Animals↗
PubMed2000

Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance.

Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase. Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase). Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains. A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range. Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases. The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains. These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins. It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.

Animals↗
PubMed1999

ATP-Binding site of annexin VI characterized by photochemical release of nucleotide and infrared difference spectroscopy.

Structural changes induced by nucleotide binding to porcine liver annexin VI (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). Photorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI characterized by reproducible changes in the amide I region. The magnitude of the infrared change was comparable to RIDS of other ATP-binding proteins, such as Ca(2+)-ATPase and creatine and arginine kinases. Analysis of RIDS revealed the existence of ATP-binding site(s) (K(d) < 1 microM) within the AnxVI molecule, comprising five to six amino acid residues located in the C-terminal portion of the protein molecule. The binding stoichiometry of ATP:AnxVI was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced changes in protein secondary structure reflected by a 5% decrease in alpha-helix content of the protein in favor of unordered structure. Such changes may influence the affinity of AnxVI for Ca2+ and modulate its interaction with membranes.

Adenosine Triphosphate↗
PubMed1999

Annexin VI interacts with adenine nucleotides and their analogs.

Annexin VI (AnxVI), a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to interact in vitro with adenine nucleotides. Furthermore, it has been proposed that within the AnxVI molecule a nucleotidde-binding domain exists, which is located in the C-terminal half of the protein, in the vicinity of Trp343. By comparison of exposure of tryptophan and multiple tyrosine residues upon nucleotide binding, as revealed by quenching of intrinsic fluorescence of AnxVI by ATP, ADP or cAMP, it can be concluded that the binding of nucleotides evokes changes in the protein tertiary structure. Moreover, in the course of present study we have found that AnxVI binds to a non-hydrolysable analog of ATP, the triazine dye Cibacron blue 3GA (CB3GA), immobilized on agarose. Binding reveals negative cooperativity with respect to protein concentration and is Ca2+-dependent. Binding is prevented by ATP. CB3GA binds to AnxVI also in solution, evoking the formation of annexin multimers. On the basis of this observation it can be suggested that interaction of CB3GA with AnxVI is useful to examine, with some limitations, the self-association of annexin molecules implying to play a role in interacting of AnxVI with biological membranes.

Adenine Nucleotides↗
PubMed1999

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.

Annexin VI (AnxVI) from porcine liver, a member of the annexin family of Ca(2+)- and membrane-binding proteins, has been shown to bind ATP in vitro with a K(d) 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 Ca(2+)-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.

Adenosine Triphosphate↗
PubMed1999

Annexin VI: an intracellular target for ATP.

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.

Adenosine Triphosphate↗
PubMed1998

Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes.

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types. This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone-induced apoptosis in rat thymocytes. It also presents annexin VI-FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin-treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time-course of membrane changes are different from those observed during dexamethasone-induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis.

Animals↗
PubMed1998

Modulation of annexin VI--driven aggregation of phosphatidylserine liposomes by ATP.

Annexin (Anx) VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. In addition, AnxVI of porcine liver is an ATP-binding protein, and ATP in vitro modulates its interaction with membranes and cytoskeletal elements (Bandorowicz-Pikuła and Awasthi, FEBS Lett. 409 (1997) 300-306). In this study, we examined the effect of ATP on phosphatidylserine (PtdSer) aggregation in the presence of annexin and on calcium-dependent binding of protein to liposomes, and found that ATP stimulates the former process, although it increases the calcium concentration necessary for half-maximal binding of AnxVI to membranes. These results were corroborated by the experiments with fluorescent analog of ATP, in which binding of ATP to AnxVI was affected by binding of Ca2+ and/or phospholipids to the protein. Taken together they favour an idea of ATP being a functional ligand for AnxVI, which even in the relative absence of Ca2+ may modulate interaction of AnxVI with PtdSer-enriched membranes.

Adenosine Triphosphate↗
PubMed1998

A nucleotide-binding domain of porcine liver annexin VI. Proteolysis of annexin VI labelled with 8-azido-ATP, purification by affinity chromatography on ATP-agarose, and fluorescence studies.

Porcine liver annexin VI (AnxVI) of Mr 68.000 is an ATP-binding protein as evidenced by specific and saturable UV-dependent labelling with 8-azido-[gamma-32P]ATP or the fluorescent analog of ATP, 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine triphosphate and by binding of AnxVI to ATP-agarose. These characteristics of purified AnxVI were used to identify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited proteolysis and the proteolytic fragments of AnxVI that retained the covalently-bound nucleotide were separated by means of gel electrophoresis and visualized by exposure of the gel to a phosphor storage screen. It was found that the AnxVI proteolytic fragments of Mr 34-36.000 and smaller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-agarose column protein fragments of similar Mr to these labelled with 8-azido-ATP were identified. The extent of AnxVI labelling with 8-azido-ATP and the distribution of proteolytic fragments varied upon calcium concentration. These results lead to the conclusion that there is a nucleotide-binding domain within the AnxVI molecule that is functionally similar to the nucleotide-binding domains of other nucleotide-binding proteins. The nucleotide-binding domain is located close to the tryptophan residue 343 of AnxVI and in close vicinity to the Ca2+- and phospholipid-binding sites of the protein. This is confirmed by the observation that the tryptophan fluorescence intensity of AnxVI decreases in the presence of a fluorescence analog of ATP in a calcium-dependent manner, due to the quenching properties of the nucleotide and/or fluorescence energy transfer from AnxVI tryptophan to fluorophore. Both processes were modulated by the presence of phospholipid molecules.

Adenosine Triphosphate↗
PubMed1998

Annexins and ATP in membrane traffic: a comparison with membrane fusion machinery.

Annexins, calcium- and membrane-binding multifunctional proteins, have been implicated in N-ethylmaleimide (NEM)-independent fusion of vesicular structures involved in membrane traffic. This view is based on intracellular localization of annexins, which are frequently associated with endosomes, chromaffin granules, caveolae, clathrin-coated pits, and other membrane compartments, engaged in endo- and exocytosis. Moreover, annexins were found to modulate budding and aggregation of vesicle membranes, to interact with cytoskeletal proteins, and, upon binding to membranes, to change the structure of lipid bilayer, leading to membrane fusion. In addition, some annexins are substrates for various protein kinases and, in membrane-bound form, reveal calcium channel activity. Recently, annexins were observed to interact in vitro and in vivo with nucleotides, ATP, GTP or cAMP, which are potent mediators of membrane traffic processes. In addition, annexin VII showed hydrolytic activity towards GTP, and similarities in the mechanism of action to that of small GTP-binding proteins were found. The aim of the present review is to summarize the observations implying annexins as possible effectors in endo- and exocytosis and to compare them with well known complexes of cytosolic and membrane proteins forming the true membrane fusion machinery within a cell, conserved from yeast to the neurons of humans.

Adenosine Triphosphate↗
PubMed1998

Adenosine 5'-triphosphate--a new regulator of annexin function. A hypothesis.

The paradigm of annexins as phospholipid-binding proteins interacting with membranes in a calcium-dependent manner has been recently questioned in light of observations that some annexin isoforms may behave like membrane integral proteins or remain associated with their target membranes at low, resting, concentrations of Ca2+ in the cytoplasm. In addition, an evidence has been presented that some annexins (annexins I, VI and VII) bind in vitro ATP and GTP, and upon binding the nucleotide the in vitro activity of these proteins is modified. However, annexins do not contain Walker A and B consensus sequences for ATP/GTP binding. This review presents the hypothesis that a new ATP-binding motif exists within the annexin molecules and that ATP may play a role of functional ligand for annexins also in vivo.

Adenosine Triphosphate↗
PubMed1997

Fluorescence spectroscopic studies on interactions between liver annexin VI and nucleotides--a possible role for a tryptophan residue.

Annexin VI is a 68-kDa calcium-, phospholipid-, and cytoskeletal-element-binding protein, which has been implicated in various processes, including calcium release and sequestration in calcifying cartilage, in a receptor-mediated endocytosis in human fibroblasts, and in secretion from chromaffin granules. In these processes it was found that, in addition to Ca2+ and annexin, the presence of ATP is also a prerequisite. In the present report we show that annexin VI binds ATP and the binding of nucleotide to protein is accompanied by quenching of an intrinsic fluorescence of annexin VI, which was found to be specific for 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, GTP and ATP, and dependent on the annexin conformation. The nucleotide-binding site within an annexin VI molecule is likely to be close to the tryptophan-containing domain of annexin VI. We propose that ATP plays the role of a physiological ligand for annexin VI, and its binding to annexin VI may represent an alternative cellular mechanism for the regulation of annexin-membrane interactions coupled to overall energy transitions in the cell.

Acrylamide↗
PubMed1997

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.

Annexin VI from porcine liver can be photoaffinity-labeled with 8-azido-[gamma-32P]ATP in a concentration-dependent, saturable manner. The extent of labeling varied with the concentration of calcium. The dissociation constant for the nucleotide was found to be in the range reported for ATP-binding proteins. The ATP analog, 2'-(or 3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate, also bound to AnxVI, as indicated by shift in its fluorescence spectra in the presence of protein. Any significant 8-azido-ATP or TNP-ATP binding was not observed with AnxIV. ATP modulated the binding of AnxVI to erythrocyte membrane and increased the Ca2+ concentration required for half-maximal binding of AnxVI to F-actin.

Actins↗