UNIPROT:P08758 - FACTA Search

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Query: UNIPROT:P08758 (annexin V)
9,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calcifiable proteolipids present in mineralizing tissues have been postulated to enhance apatite deposition by structuring membrane phosphatidylserine molecules into a conformation conducive to mineral formation. To examine whether proteolipid-like molecules are present in mineralizable matrix vesicles (MV), the vesicles were first extracted with chloroform/methanol (2:1, v/v), and then with chloroform/methanol/HCl (200:100:1, v/v) and the organic-soluble proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Protein fractions were analyzed by Coomassie Blue staining and by immunoblot analysis of electrophoretically transferred MV protein with antisera to the 33- and 36-kDa annexins. We found that several MV proteins selectively partitioned into the lipophilic milieu under acidic conditions; however, very little protein did so at neutral pH. The principal organic-soluble MV proteins had molecular masses of 14, 33, and 36 kDa, with lesser bands at 28, 30, and 68 kDa. Immunological analyses revealed that the 33- and 36-kDa proteins were the MV annexins; the 14-kDa protein appeared to be hemoglobin, based on NH2-terminal sequencing. Our findings indicate that under acidic conditions the 33- and 36-kDa MV annexins undergo a conformational change which imparts a marked increase in the hydrophobicity of the proteins. While these observations reveal that the annexins possess proteolipid-like properties, radiolabeling and immunoprecipitation studies using [3H]myristic acid in chondrocyte cultures indicate that the MV annexins are not myristylated. Amino-terminal sequence analysis of the peptides generated by site-specific cleavage of the 33- and the 36-kDa MV annexins at tryptophan residues indicate that the 33 kDa is highly homologous to anchorin CII, a protein known to bind type II collagen, while the 36-kDa protein shares close homology with endonexin II, a tyrosine kinase substrate.
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PMID:Matrix vesicle annexins exhibit proteolipid-like properties. Selective partitioning into lipophilic solvents under acidic conditions. 203 7

The annexins are a novel class of calcium-dependent membrane binding proteins with highly homologous sequences and similar binding characteristics. In order to better define structural parameters of the membrane-bound form, the localization of tryptophan residues in several of these proteins was studied by use of quenchers of their intrinsic fluorescence. Lipocortin I contains a single tryptophan located near its N-terminus, while the single tryptophan in lipocortin V is located in a repeated consensus sequence. Calcium-dependent binding to vesicles composed of 50% egg phosphatidylcholine and 50% bovine brain phosphatidylserine was accompanied by an increase in emission intensity resulting from a relief of internal quenching. The tryptophan fluorescence of bound lipocortin I was nearly unaffected by substituting the quencher 1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphocholine (5-PC) for egg phosphatidylcholine, while that of the lipocortin V tryptophan was quenched significantly. With the quenching doxyl spin-label located deeper in the bilayer at the 12- and 16-positions of the acyl chain, the quenching was progressively weaker, suggesting an interfacial location for the tryptophan of lipocortin V. The same experiments with lipocortin I show almost no quenching in any case, suggesting that this tryptophan near the amino terminus is protected or oriented away from the membrane surface. Data on the bovine liver calelectrins are also presented showing that endonexin also has a tryptophan residue that interacts strongly with phospholipids.
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PMID:Location of tryptophans in membrane-bound annexins. 213 93

We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-beta-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.
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PMID:Collagen binding activity of recombinant and N-terminally modified annexin V (anchorin CII). 767 28

Annexins V and VI are two members of the annexin protein family, each of which associate with phospholipid vesicles in a calcium-dependent manner. They may be important intracellular calcium response elements. Titration calorimetry and spectroscopic techniques showed striking features of these interactions. For annexin V and VI, the total heat release from calcium-dependent binding to bilayer vesicles composed of phosphatidylserine/phosphatidylcholine was -25 and -38 kcal/mol, respectively. The enthalpy of association (delta Hassoc) for the respective protein-calcium interactions was about -11 and -5 kcal/mol. The delta Hassoc for the annexin VI-vesicle interaction was largely independent of the headgroup of the anionic phospholipid and of the pH from 7.1 to 8.4. Use of phosphatidylethanolamine as the neutral phospholipid resulted in a slightly more negative delta Hassoc. Enthalpy was either independent of vesicle size (annexin V) or showed a slightly more negative value for large vesicles (annexin VI). The delta Hassoc for annexin VI-membrane interaction was not constant during protein titration but became more exothermic with higher protein density on the membrane. This behavior was surprising because the equilibrium constant showed negative cooperativity with respect to protein density [Bazzi, M. D., & Nelsestuen, G. L. (1991) Biochemistry 30, 7970-7977]. Apparently, entropy changes occurred which were large and negative, thereby compensating for the increasingly negative enthalpy but decreasing affinity as protein density was increased. In fact, the exothermic process coincided more closely with a change in the intrinsic tryptophan fluorescence of annexin VI than with actual protein-membrane binding. Circular dichroism detected very small changes in protein secondary structure during these events. The observed delta Hassoc for annexin-membrane interaction appeared to involve contributions from the membrane as well as from the protein. Annexin-membrane binding may exert large effects on the membrane that could serve a regulatory capacity in the cell.
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PMID:Direct enthalpy measurements of the calcium-dependent interaction of annexins V and VI with phospholipid vesicles. 794 31

The binding of calcium ions to annexin V in the absence of phospholipids has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of calcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by its "blue" fluorescence emission maximum (325 nm). This corresponds well with the description of the structure determined by X-ray crystallography of several crystal forms. The Trp187 time-resolved fluorescence decay shows the existence of a fast (picosecond) excited-state reaction which can involve the formation of an H-bond between the indole NH group and the proximate epsilon-OH and/or alpha-carbonyl groups of Thr224. Titration with calcium tends to stabilize the overall structure, as shown by circular dichroism, while leading to large modifications of the local structure around Trp187 making it accessible to the solvent as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating concentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecules of the solvation shell, is observed. These observations correlate well with the conformational change observed in crystal structures obtained in high calcium concentrations, involving the removal of Trp187 from the buried position to the surface of the molecule [Sopkova, J., Renouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321-1324]. In the solvent-exposed conformation, the indole ring becomes mobile in the subnanosecond and nanosecond time range. This conformational change and the increase in local flexibility can be important for the accommodation of the protein on the surface of phospholipid membranes.
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PMID:The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study. 816 3

Annexin V is part of a family of Ca(2+)-dependent phospholipid-binding proteins, whose purported functions are related to their interactions with biological membranes. While Ca(2+)-dependent binding to phospholipids is well-established, the specific structural interactions within the phospholipid-binding sites have only been inferred to resemble those of phospholipase A2, with no direct structural evidence. In this study, the binding avidity of various phospholipid analogs, with variations at the headgroup or sn-2 acyl chain, was monitored in a C12E8 detergent micelle system using the increase in fluorescence of tryptophan 187. Micelles also contained excess negative surface charge to saturate a nonspecific component of the binding. The Ca2+ and phospholipid concentrations required for the binding of annexin V to various phospholipid headgroups were very similar, except for the relatively weak binding to phosphatidylinositol (PI). The unique close proximity of the PI sugar ring to the phosphate group may lead to steric hindrance in this case. Binding was also strongly dependent on the presence of an sn-3 phosphate group and an sn-2 acyl chain, as previously observed. The relatively shallow nature of the annexin V phospholipid-binding sites was reflected by the nearly equivalent binding of D and L versions of phospholipids, i.e., a large shift in the position of the sn-1 acyl chain is accommodated in this case. Binding of annexin V does not specifically require an ester carbonyl oxygen, as it occurs with ether-linked, amide-linked, and phosphonate-linked sn-2 hydrocarbon chains, under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phospholipid determinants for annexin V binding sites and the role of tryptophan 187. 818 Feb 11

Annexin V is a Ca(2+)-dependent, phospholipid-binding protein that may have one or more membrane-related functions. The binding of annexin V to phospholipids in a detergent micelle matrix was studied to attempt to determine directly the stoichiometry of specific phospholipid-binding sites and the importance of negative charge. When annexin V binds to phospholipids, a large increase (severalfold) of the emission intensity of tryptophan 187 is observed. This intensity change was used to monitor the binding to phosphatidylcholine (PC) or phosphatidylserine (PS) at varying ratios with the detergent, octaethylene glycol monododecyl ether (C12E8). No binding to PC alone in these micelles could be observed, while approximately 10 PS molecules per micelle were required to observe binding. However, inclusion of negatively charged amphiphiles in the micelles, such as oleic acid or dodecyl sulfate, allowed the observation of binding to PC and decreased the number of phospholipids per micelle necessary for binding to both PS and PC. By including increasing proportions of dodecyl sulfate in the C12E8 micelles, a minimum average number of PS or PC per micelle of approximately 3-4 was required for complete binding. Labeling with photoreactive phospholipids under similar conditions led to an average of approximately 4-5 phospholipids covalently bound per annexin V monomer. Since annexin V has four similar domains, it is reasonable to suggest that one phospholipid binding site is associated with each domain, although as few as three functional domains may be sufficient for binding. Efficient binding required certain structural features of the phospholipid, including a phosphate group, an sn-2 acyl chain, and at least a few carbons on the sn-2 chain. Phospholipid headgroups were almost irrelevant, except for important surface charge effects on the interfacial ionic double layer. A negative surface charge on the micellar aggregate nonspecifically increases the Ca2+ concentration near the micelle surface and may also directly enhance the affinity of annexin V for phospholipids, as shown by the decreased two-dimensional phospholipid concentration necessary for binding. The ability to bind to zwitterionic phospholipids in the presence of a nonspecific negative surface charge may be relevant to the extracellular functions of this protein. Relatively weak individual phospholipid-binding sites that easily exchange were observed, suggesting rapid exchange of phospholipids between the sites on membrane-bound annexin V. These data suggest a working hypothesis that includes approximately four binding sites specific for phospholipid phosphate groups and sn-2 acyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calcium-dependent annexin V binding to phospholipids: stoichiometry, specificity, and the role of negative charge. 821 40

Annexins are a family of calcium- and phospholipid-binding proteins implicated in mediating membrane-related processes such as secretion, signal transduction, and ion channel activity. The crystal structure of rat annexin V was solved to 1.9 angstrom resolution by multiple isomorphous replacement. Unlike previously solved annexin V structures, all four domains bound calcium in this structure. Calcium binding in the third domain induced a large relocation of the calcium-binding loop regions, exposing the single tryptophan residue to the solvent. These alterations in annexin V suggest a role for domain 3 in calcium-triggered interaction with phospholipid membranes.
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PMID:Rat annexin V crystal structure: Ca(2+)-induced conformational changes. 836 44

Annexin V is a Ca(2+)-dependent phospholipid-binding protein that may have one or more membrane-related functions, including inhibition of blood coagulation. The fluorescence of the single tryptophan of annexin V was used to monitor Ca2+ and/or phospholipid binding in terms of emission wavelength, emission intensity, and susceptibility to acrylamide quenching. In the absence of phospholipid, Ca2+ titration showed a strong red shift of the wavelength of maximal emission to approximately 345 nm, where a small increase in intensity occurred and was half maximal at approximately 3 mM Ca2+. The Stern-Volmer quenching constant due to acrylamide was only 5.2 M-1 for annexin V alone, indicating limited aqueous exposure of the tryptophan, but 36 M-1 for a Ca(2+)-bound form, indicating full exposure. Binding to both negatively charged and zwitterionic phospholipids was accompanied by a very large increase in fluorescence emission intensity, a red shift, and low exposure to acrylamide. Calculated concentrations of Ca2+ near the surface of negatively charged vesicles suggested that the exposure of tryptophan by Ca2+ binding to annexin V was sufficient for binding of the protein to all vesicles tested, including those composed of oleic acid and phosphatidylcholine (PC), but not to those composed of pure PC. When binding to PC was monitored, the phenomena associated with phospholipid binding were observed separately, at higher Ca2+ concentration, from the red shift and the high exposure to acrylamide due to Ca2+ binding alone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Relationship between annexin V tryptophan exposure, calcium, and phospholipid binding. 849 44

The structure of recombinant human annexin III was solved to 1.8 A resolution. Though homologous to annexin I and V, the annexin III structure shows significant differences. The tryptophan in the calcium loop of the third domain is exposed to the solvent, as in the structure of annexin V crystallized in high calcium concentrations, although the annexin III crystals were prepared at low calcium concentrations. The position of domain III relative to the other domains is different from both annexin V and I, suggesting further flexibility of the molecule. The entire N-terminus of the protein is well-defined in the present structure. The side chain of tryptophan 5 interacts with the hinge region of the hydrophillic channel, which could have an effect on the potential mobility of this region, as well as on its possible calcium channel behavior.
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PMID:The high-resolution crystal structure of human annexin III shows subtle differences with annexin V. 863 53

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