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A new strategy for sensitive detection of early stage apoptosis was proposed based on silver-enhanced gold nanoparticle (GNP) label method. Annexin-V modified substrate was constructed via layer-by-layer (LBL) method for specific capture of early stage apoptotic Jurkat cells. A new kind of aminophenylboronic acid modified gold nanoparticle (APBA-GNP) was synthesized and utilized for labeling cells, followed by silver enhancement. Anodic stripping voltammetry (ASV) was applied to sensitive detection of Ag(+) dissolved from the deposited silver particles, which reflected the number of cells. A good linear range from 1 × 10(2) to 3.5 × 10(3) cells was achieved, with a detection limit of 38 apoptotic cells. Moreover, the gray color of silver enhancement could be observed by the naked eye, which could be used to tell apoptotic cells apart from normal cells. Therefore, using the silver-enhanced GNP label method, apoptotic cells could not only be sensitively detected via electrochemical technique, but also can be discriminated from normal cells by the naked eye., (© 2013 Elsevier B.V. All rights reserved.)

This paper focuses on the targeting of single-walled carbon nanotubes (SWNTs) for the treatment of breast cancer with minimal side effects using photothermal therapy. The human protein annexin V (AV) binds specifically to anionic phospholipids expressed externally on the surface of tumour cells and endothelial cells that line the tumour vasculature. A 2 h incubation of the SWNT-AV conjugate with proliferating endothelial cells followed by washing and near-infrared (NIR) irradiation at a wavelength of 980 nm was enough to induce significant cell death; there was no significant cell death with irradiation or the conjugate alone. Administration of the same conjugate i.v. in BALB/c female mice with implanted 4T1 murine mammary at a dose of 0.8 mg SWNT kg(-1) and followed one day later by NIR irradiation of the tumour at a wavelength of 980 nm led to complete disappearance of implanted 4T1 mouse mammary tumours for the majority of the animals by 11 days since the irradiation. The combination of the photothermal therapy with the immunoadjuvant cyclophosphamide resulted in increased survival. The in vivo results suggest the SWNT-AV/NIR treatment is a promising approach to treat breast cancer.

In contrast to normal healing wounds, chronic wounds commonly show disturbances in proteins regulating wound healing processes, particularly those involved in cell proliferation and protein degradation. Multidimensional protein identification technology MS/MS was conducted to investigate and compare the protein composition of chronic diabetic foot exudates to exudates from split-skin donor sites of burn victims otherwise healthy. Spectral counting revealed 188 proteins differentially expressed (more than twofold and p-value <0.05) in chronic wounds. Most were involved in biological processes including inflammation, angiogenesis, and cell mortality. Increased expression of the inflammatory response stimulating S100 proteins, predominantly S100A8 and S100A9 (almost tenfold), was identified. Matrix metalloproteinases (MMPs) MMP1, MMP2, and MMP8 were identified to be elevated in chronic wounds with significant impact on collagen degradation and tissue destruction. Further, proteins with antiangiogenic properties were found at higher expression levels in chronic wounds. Reduced angiogenesis leads to drastic shortage in nutrition supply and causes increased cell death, demonstrated by Annexin A5 exclusively found in chronic wound exudates. However, excessive nucleic and cytosolic material infers cell death occurring not only by apoptosis but also by necrosis. In conclusion, mass spectrometric investigation of exudates from chronic wounds demonstrated dramatic impairment in wound repair with excessive inflammation, antiangiogenic environment, and accelerated cell death., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell death by apoptosis has been studied for many years using fluorescently labeled annexin V. Annexin V shows high affinity for the phosphatidylserine that becomes enriched in the outer leaflet of the plasma membrane during apoptosis, but not necrosis, allowing differentiation between the two types of cell death. In this chapter we detail two methods for the purification of annexin V. The first is an untagged recombinant protein using a three step Fast Protein Liquid Chromatography (FPLC) method, and the second using a single step purification protocol via a glutathione S-transferase (GST) tag. Labeling of the resulting annexin V with a fluorescent dye to allow visualization of the protein is also explained. Finally, two methods are described in which a fluorescently labeled derivative of annexin V is used to detect apoptosis, namely the in vitro method of fluorescence-activated cell sorting (FACS) where fluorescent annexin V is used to differentiate apoptotic and necrotic cells within a population; and detection of apoptosing retinal cells (DARC) allowing the identification of apoptotic cells in the retina in vivo.

An endogenous inhibitor of L-Dopa decarboxylase activity was identified and purified from human placenta. The endogenous inhibitor of L-Dopa decarboxylase (Ddc) was localized in the membrane fraction of placental tissue. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K did not affect membrane-associated Ddc inhibitory activity, suggesting that a population of the inhibitor is embedded within membranes. Purification was achieved by extraction from a nondenaturing polyacrylamide gel. The purification scheme resulted in the isolation of a single 35 kDa band, bearing L-Dopa decarboxylase inhibitory activity. The purified inhibitor was identified as Annexin V. The elucidation of the biological importance of the presence of an L-Dopa decarboxylase activity inhibitor in normal human tissues could provide us with new information leading to the better understanding of the biological pathways that Ddc is involved in.

Apoptosis is a mode of programmed cell death that is widely used to eliminate cells during development, tissue homeostasis, infection or in response to injury. Alterations to the plasma membranes of apoptotic cells trigger recognition and engulfment of such cells by phagocytes. Measurement of plasma membrane phosphatidylserine externalization, using fluorescently labeled annexin V, is widely used for the detection of apoptotic cells. Here we describe protocols for bacterial expression, purification and FITC labeling of recombinant annexin V. By following the method outlined in this protocol, it is possible to produce milligram amounts of recombinant annexin V within 3 d. We also describe a method for the assessment of annexin V binding to cell populations by flow cytometry or fluorescence microscopy.

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.

Apoptosis is a mode of cell death that is accompanied by specific alterations to the plasma membrane that promote the recognition and engulfment of these cells by phagocytes. Although several such membrane alterations have been defined, redistribution of phosphatidylserine from the inner to the outer plasma membrane leaflet has become one of the most widely used markers for apoptotic cells in mammals. This is largely due to the availability of a sensitive and specific probe for this event in the form of the phosphatidylserine-binding protein, annexin V. Here, we describe methods for the expression and purification of recombinant polyhistidine-tagged annexin V from Escherichia coli. Recombinant annexin V is highly soluble and is thus readily expressed and purified to high yields; typically in the region of 4microg of protein per ml of bacterial culture. We also describe methods for conjugation of this protein to the FITC fluorophore and for its use for the detection of apoptotic cells by flow cytometry or fluorescence microscopy.

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.

Dicalcin (renamed from p26olf) is a dimer form of S100 proteins found in frog olfactory epithelium. S100 proteins form a group of EF-hand Ca(2+)-binding proteins, and are known to interact with many kinds of target protein to modify their activities. To determine the role of dicalcin in the olfactory epithelium, we identified its binding proteins. Several proteins in frog olfactory epithelium were found to bind to dicalcin in a Ca(2+)-dependent manner. Among them, 38 kDa and 35 kDa proteins were most abundant. Our analysis showed that these were a mixture of annexin A1, annexin A2 and annexin A5. Immunohistochemical analysis showed that dicalcin and all of these three subtypes of annexin colocalize in the olfactory cilia. Dicalcin was found to be present in a quantity almost sufficient to bind all of these annexins. Colocalization of dicalcin and the three subtypes of annexin was also observed in the frog respiratory cilia. Dicalcin facilitated Ca(2+)-dependent liposome aggregation caused by annexin A1 or annexin A2, and this facilitation was additive when both annexin A1 and annexin A2 were present. In this facilitation effect, the effective Ca(2+) concentrations were different between annexin A1 and annexin A2, and therefore the dicalcin-annexin system in frog olfactory and respiratory cilia can cover a wide range of Ca(2+) concentrations. These results suggested that this system is associated with abnormal increases in the Ca(2+) concentration in the olfactory and other motile cilia.

Biochemical studies have suggested that annexin 2 (A2) may participate in cytomegalovirus (CMV) infection. In the current work, effects of A2 monomer (p36) and heterotetramer (A2t; p36(2)p11(2)) were investigated. Demonstrating a role for endogenous A2, the four stages of infection that were followed were each inhibited by anti-p36 or anti-p11 at 37 degrees C. Immuno-inhibition was attenuated when the virus and cells were pre-incubated at 4 degrees C to coordinate virus entry initiated afterwards at 37 degrees C, reconciling controversy in the literature. As an explanation, CMV-induced phosphorylation of p36 was prevented by the 4 degrees C treatment. Supporting these immuno-inhibition data, purified A2t or p11 increased CMV infectious-progeny generation and CMV gene expression. A specific role for A2t was indicated by purified p36 having no effect. Unlike other steps, primary plaque formation was not enhanced by purified A2t or p11, possibly because of undetectable phosphorylation. As annexins 1 (A1) and 5 (A5) interact with A2, their effect on CMV was also tested. Both purified proteins inhibited CMV infection. In each experiment, the concentration of A1 required for half-maximal inhibition was five- to 10-fold lower than that of A5. Addition of A2 opposed A1- or A5-mediated inhibition of CMV, as did certain A2-specific antibodies that had no effect in the absence of added A1 or A5. Transfection of the p36-deficient cell line HepG2 increased CMV infection and was required for inhibition by the other annexins. These data suggest that CMV exploits A2t at physiological temperature to oppose the protection of cells conferred by A1 or A5.

Due to their involvement in a variety of physiological and pathological processes, different isoforms of annexins are being utilized as markers of some human diseases and bio-imaging of tissue injury (due to apoptosis), and have been proposed as drug delivery vehicles. These, in addition to extensive biophysical studies on the role of annexins in organizing lipid domains in biological membranes, have necessitated development of an efficient protocol for producing annexins in bulk quantities. In this paper, we report a one-step purification protocol for annexin a5 without using lipid vesicles or involving any column chromatographic step. Depending on the growth and expression condition, a fraction of recombinant annexin a5 (cloned in pET3d vector) was sequestered into inclusion bodies. When these inclusion bodies were dissolved in 6 M urea, subjected to a 10-fold snap dilution in the presence of 5 mM Ca(2+) and stored overnight at 4 degrees C, annexin a5 was precipitated as a homogenous protein as judged by SDS-PAGE. This one-step purification protocol produced about 35 mg of highly purified annexin a5 per liter of bacterial culture. The annexin a5 purified from inclusion bodies exhibited similar properties to that obtained from the soluble fraction using the conventional lipid-partitioning approach. Our purification protocol for annexin a5 elaborated herein is equally effective for purification of annexin A2, and we believe, will serve as general protocol for purifying other annexins in bulk quantities for diagnostic as well as detailed biophysical studies.

Recombinant human annexin A5 (rh-annexin A5) was originally used to detect early stages of apoptosis in vitro. With the development of radioactive labeling and imaging techniques, annexin A5 labeled with radioactive markers can play a more important role in monitoring apoptotic cells in vivo. To obtain highly pure rh-annexin A5 with an easy and inexpensive purification approach, we constructed a pJLA503-annexin A5 expression plasmid, which could overexpress human annexin A5 in a soluble form in Escherichia coli. Then a novel purification method based on Ca2+-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of rh-annexin A5 was increased to 98%. To confirm the PS affinity of rh-annexin A5 produced by this purification protocol, a simple and reliable lipid membrane model was prepared and used in the binding test. As a probe to detect apoptosis, the fluorescein isothiocyanate-labeled rh-annexin A5 was incubated with apoptotic cells. The results showed that the labeled rh-annexin A5 possessed high affinity for PS molecule and was able to indicate different apoptotic states.

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.

The anionic phospholipid, phosphatidyl-L-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid beta-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor-initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents.

A 35 kDa protein was purified from rat spinal ganglia and sensory fibers. Combined direct trypsin digest and liquid chromatography ion trap mass spectrometry analysis, the 35 kDa protein was identified as annexin V. We then studied the distribution of serum antibodies to annexin V in patients with peripheral neuropathy. We found serum positive antibodies to annexin V only in some patients with immune-mediated neuropathy. This indicated that humoral immune responses to annexin V might play a role in the pathogenesis of autoimmune sensory neuropathy or sensory neuronopathy.

A human Annexin V-Hirudin chimeric protein, Annexin V-Hirudin C, was expressed in Escherichia coli. A broad range of parameters such as plasmid stability during propogation and expression, expression capacity stability, the culture media, the growth time before induction and the induction duration were examined and optimized. Recombinant Annexin V-Hirudin C was purified from the cell lysate supernatants by ethanol precipitation, DEAE-cellulose chromatography and Sephadex G-75 chromatography, and the purified protein showed dose-dependent thrombin inhibitory activity. The overall production of purified Annexin V-Hirudin C protein is 10 mg/l/OD600.

A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE.

Bovine lactadherin binds to the alpha(v)beta(3) and alpha(v)beta(5) integrins in an RGD-dependent manner and also to anionic phospholipids. During the affinity purification of lactadherin binding receptors, a 35-kDa protein persistently coeluted with the alpha(v)beta(5) integrin receptor. Subsequently, peptide mapping, amino acid sequencing, and mass spectrometry analysis identified this protein as bovine annexin-V. Annexin-V accompanied the integrin receptor eluted with either RGD peptide or with EDTA suggesting that annexin-V bound specifically to the alpha(v)beta(5) integrin. To further investigate this putative interaction of annexin-V with the alpha(v)beta(5) integrin receptor, human annexin-V and intracellular domains of the human alpha(v)beta(5) integrin subunits were used in ligand blotting assays. Radiolabeled annexin-V showed weak binding to the intracellular part of beta(5) integrin subunit. However, by adding the aminophospholipid, phosphatidyl serine, the interaction with the beta(5) cytoplasmic peptide was enhanced many fold. Furthermore, the interaction was shown to be independent of phosphorylation, as annexin-V bound to unphosphorylated beta(5) peptide at a similar level to the phosphorylated peptide. Since binding of annexin-V to the alpha(v) integrin subunit tail was not detected, annexin-V was shown to associate specifically with the beta(5) cytoplasmic tail. Together these findings suggest a novel link between annexins and the integrin receptor family., ((c)2002 Elsevier Science (USA).)

A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a K(d) of 200 microM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.

Human annexin V cDNA was cloned into plasmid pET19b and fused to a ten consecutive histidine tag at N-terminal. When expressed in E. coli BL21(DE3) LysS, the recombinant His10-annexin V accumulated in soluble form in the cytoplasm. By two-step chromatography, i.e., metal chelate affinity chromatography and anion exchange chromatography, recombinant His10-annexin V was purified to homogeneity on silver-stained SDS-PAGE gel. Recombinant annexin V, 7.4 mg, was obtained from a 1 litre flask culture.

Annexin V is a member of a family of structurally homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. The structure of the soluble form of annexin V has been solved by X-ray crystallography, while electron crystallography of two-dimensional (2D) crystals has been used to reveal the structure of its membrane-bound form. Two 2D crystal forms of annexin V have been reported to date, with either p6 or p3 symmetry. Atomic force microscopy has previously been used to investigate the growth and the topography of the p6 crystal form on supported phospholipid bilayers (Reviakine et al., 1998). The surface structure of the second crystal form, p3, is presented in this study, along with an improved topographic map of the p6 crystal form. The observed topography is correlated with the structure determined by X-ray crystallography., (Copyright 2000 Academic Press.)

CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death., (Copyright 2000 Academic Press.)

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.

After isolation of rabbit skeletal muscle membranes in the presence of Ba2+ or Ca2+, significant portions of annexin V and VI tightly bind to membranes and become inaccessible for chelating agents. Tightly bound annexin VI is virtually completely solubilized only after treatment with a buffer supplemented both with EGTA and detergent. The portion of tightly bound annexin V cannot be removed even by extraction with buffer containing both EGTA and detergent. In some cases, tightly bound annexin V or VI is detected even in the control (not treated with cations) membranes, thus indicating the possible formation of tight annexin--membrane complexes in situ. The addition of exogenous cations seems to promote only the accumulation of tightly bound annexins within the cell. After temperature-induced phase separation, annexin V and VI bound to the membranes isolated in the presence of Ba2+ or Ca2+ remains mainly in the aqueous phase, similarly to annexins isolated from the control membranes. Neither annexin partitions into the detergent-enriched phase. This indicates the absence of hydrophobicity change in comparison with the standard EGTA-soluble annexins.

Mouse annexin V genomic clones were characterized by restriction analysis, Southern blotting and DNA sequencing. The entire gene spans close to 50 kb of the mouse genome and contains 14 exons ranging in size from 31 bp for exon 2 to 482 bp for exon 13 up to the polyadenylation site. Intron sizes range from 111 bp for intron 1b to more than 17 kb for intron 2. Non-coding exon 1 is present in two alternative forms separated by approx. 7.4 kb, and the two promoters associated with exons 1a and 1b are quite distinct. The upstream promoter has a TATA box and may direct the limited, tissue-specific expression of mRNA transcripts containing exon 1a. The downstream, TATA-less promoter has high G+C content, and exon 1b predominates among abundantly expressed mRNA species. The conservation of certain cis-elements, including Sp1, AP2, gamma-IRE and NF-IL6, in orthologous species of annexin V genes points to their possible role in trans-acting protein factor binding and gene regulation. Primer-extension analysis revealed multiple origins for transcription, with principal start sites 100-150 bp upstream of the ATG start codon in exon 2. Intron 4 was longer than that previously identified in the orthologous rat gene due to the integration of an apparently complete copy of the murine endogenous retrovirus element, MuERV-L. Phylogenetic analysis of annexin V from 12 species and the presence of neighbouring loci with paralogous counterparts linked to annexin VI pointed to the common ancestry of these genes via chromosomal duplication more than 600 million years ago.

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.

In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.

The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.

Annexin V is a major component of matrix vesicles and has a role in mediating the influx of Ca2+ into these vesicles, thus promoting the initiation of hypertrophic cartilage matrix mineralization. However, the mechanisms and factors regulating annexin V-mediated Ca2+ influx into these vesicles are not well understood. Since the lipid composition of matrix vesicles differs from that of the plasma membrane of chondrocytes and is rich in phosphatidylserine, we asked whether the lipid composition may regulate annexin V function. We prepared liposomes containing different concentrations of phosphatidylserine and determined how the lipid composition affected (a) the interactions between annexin V and liposomes and (b) annexin V-mediated Ca2+ influx into the liposomes. We found that annexin V was able to bind to every liposome tested. However, we observed the most prominent increases in tryptophan 187 emission intensity, a measure of the degree of interactions between annexin V and lipid bilayers, only with liposomes containing a high concentration of phosphatidylserine. In addition, a significant fraction of annexin V associated with phosphatidylserine-rich liposomes was not extractable by EDTA treatment but required a detergent, indicating that annexin V inserts into bilayers of these liposomes. Chemical cross-linking analysis revealed that matrix vesicles and phosphatidylserine-rich liposomes induced the formation of the annexin V hexamer. Interestingly, a significant Ca2+ influx in the presence of annexin V occurred only in liposomes containing a high phosphatidylserine content. Moreover, annexin V-mediated Ca2+ influx into these liposomes was inhibited (i) by anti-annexin V antibodies and (ii) by treatment with zinc and cadmium, indicating the essential role of the protein in Ca2+ influx. The results of this study indicate that phosphatidylserine-rich bilayers induce the formation of a hexameric annexin V, possibly leading to a Ca2+-dependent insertion of annexin V into the bilayer and establishment of annexin V-mediated Ca2+ influx into matrix vesicles or liposomes. The phosphatidylserine-rich membrane of matrix vesicles in vivo may thus offer an ideal specialized environment in which the biological function of annexin V is optimized, leading to rapid Ca2+ influx, intralumenal crystal growth, and cartilage matrix mineralization.

To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV). The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris. Transformants were monitored for the secretion of fibrinolytic activity. The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions. It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence. Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis. Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis. Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium. The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin. The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation. Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV.

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.

Fractionation of internal matrix proteins from chicken liver nuclei led to the isolation of a 32 kDa protein which was identified by partial amino acid sequence and immunological analysis as annexin V, an unreported nuclear matrix component. Our results showed that this protein is preferentially associated with the internal nuclear matrix fraction, since this is the only nuclear fraction where the protein can be immunodetected. Immunostaining on cultured cells also revealed a nuclear distribution with the exclusion of the nucleolar compartment and an association with cytosolic filamentous structures most likely corresponding to the cytoskeleton. Moreover, immunostaining on extracted cells to reveal the nuclear matrix showed a network-like distribution. Since annexin V has been reported as an inhibitor of protein kinase C, its nuclear localization in association with the internal matrix, which plays an important role in several nuclear processes, indicates its involvement in the regulation of signal transduction.

Maintenance of renal function in liver cirrhosis requires increased synthesis of arachidonic acid derived prostaglandin metabolites. Arachidonate metabolites have been reported to be involved in modulation of liver damage. The purpose of the present study was to establish whether the first enzyme of the prostaglandin cascade synthesis, the phospholipase A2(PLA2) is altered in liver cirrhosis induced by bile duct excision. The mRNA of PLA2(group I and II) and annexin-I a presumptive inhibitor of PLA2 enzyme was measured by PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. The mean mRNA ratio of group II PLA2/GAPDH was increased in liver tissue by 126% (P < 0.001) and in kidney tissue by 263% (P < 0.006) following induction of liver cirrhosis. The increase in group II PLA2 mRNA in cirrhotic animals was reflected by an increase in PLA2 protein and enzyme activity in both liver and kidney tissues. Since the mRNA of group I PLA2 was not detectable and Group IV PLA2 activity measured in liver and kidney tissue samples was very low and not changed following induction of cirrhosis, it is likely that the major PLA2 activity measured in liver and kidney corresponds to group II PLA2 enzyme. The mean mRNA ratio of annexin-I/GAPDH was increased in liver tissue by 115% (P < 0.05) but unchanged in kidney tissue following induction of cirrhosis. The protein content of annexin-I and -V were not affected by bile duct excision in liver and kidney tissue indicating that upregulation of group II PLA2 activity was not due to downregulation of annexin-I or -V. Group II PLA2 activity of glomerular mesangial cells stimulated by interleukin-1 beta was enhanced by bile juice and various bile salts. In conclusion, activity of group II PLA2 is upregulated partly due to enhanced transcription and translation in cirrhosis and is furthermore augmented by elevated levels of bile salts.

Annexin V is a human protein that binds with high affinity to the abundant phosphatidylserine molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis. We designed two chimeras that use annexin V as a means to target thrombolytic agents to platelet-containing thrombi: prourokinase (1-411)-annexin V (1-320); and prourokinase (144-411)-annexin V (1-320) (amino acid numbers of parent proteins given in parentheses). Chimeras were produced by cytoplasmic expression in Escherichia coli, refolded, and purified in single-chain form. Both chimeras had the same specific activity as annexin V in binding to cell membranes containing exposed phosphatidylserine. After activation with plasmin, both chimeras had specific amidolytic activity similar to that of urokinase. Both chimeras activated plasminogen in vitro with kinetic parameters similar to those for urokinase, and both showed full activity compared to urokinase in an assay of clot lysis in vitro. This study shows the feasibility of producing chimeric plasminogen activators in which annexin V provides the thrombus-targeting component; although not yet tested in vivo, such chimeras may have advantages over antibody-based targeting agents.

Objective: The aim of this study was to characterize a 33-kDa protein (p33) isolated from bovine liver and to determine the subcellular localization of the protein in rat cardiocytes as well as in non-cardiac tissues., Methods: Cycles of calcium-induced precipitation coupled with EGTA-resolubilization were used to isolate crude annexins from bovine and rat tissues. Column chromatography was performed to purify the p33 from the crude annexins. The protein was identified as annexin V by partial amino acid sequence determination. Specificity of anti-annexin V antibody was examined by using Western blotting after one- or two-dimensional electrophoresis. For characterization of the protein, immunofluorescence microscopy and actin-binding assays were carried out., Results: Immunofluorescence microscopy showed that annexin V was stained as a striated pattern along myofibrils on frozen sections of both atria and ventricles of adult rats. The striations were seen more clearly in cultured rat atrial myocytes. Examination of doubly stained cardiocytes with anti-annexin V and anti-alpha-actinin by a confocal laser scanning microscope suggests that annexin V is localized in association with the Z-line of rat cardiac myocytes. We also found that annexin V was stained intensely in actin-rich regions of non-cardiac tissues such as bile canaliculi of rat liver and brush border-terminal web region in the epithelial cells of both small intestine and kidney proximal tubular cells. F-actin binding experiments revealed that annexin V failed to bind to F-actin directly in vitro., Conclusion: Our results suggest that annexin V is a new component of the Z-line in rat cardiocytes and might be involved in regulation of its organization.

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.

Annexins are a family of proteins with calcium- and phospholipid-binding properties. The present study was performed to identify which members of the annexin family are present in rat heart and to determine the cellular and subcellular distribution of annexin V, the most prominent annexin in rat cardiac tissue, in isolated ventricular myocytes and cultured endothelial and fibroblast-like cells. The presence of annexin I plus II, III, IV, V and VI in rat cardiac tissue was positively established with western blot analysis. Immunohistochemistry and western blot analysis revealed that annexin V is present in both cardiomyocytes and non-myocytal cells of the heart. In endothelial cells and fibroblast-like cells annexin V is predominantly localized in the cytoplasm and in cardiac myocytes in close vicinity of the sarcolemma. This last finding is confirmed by electron microscopy. Northern blot analysis demonstrated that all cell types investigated showed expression of annexin V. Annexin V mRNA levels were highest in the fibroblast-like cells, followed by the endothelial cells, and a weak signal was observed in the cardiomyocytes. By means of a sandwich-type enzyme-linked immunosorbent assay (ELISA) annexin V content in intact adult rat heart, isolated myocytes, cultured cardiac endothelial cells and fibroblast-like cells was found to be 0.70, 0.17, 1.63 and 3.84 micrograms/mg total protein, respectively. The differences in subcellular localization of annexin V in myocytes and non-myocytes suggest differences in biological function of annexin V in the various cell types.

The cloning, purification and characterization of full-length annexin V, expressed intracellularly in Saccharomyces cerevisiae is detailed. Following homogenization in a glass bead mill, clarification by ultracentrifugation and fractional ammonium sulfate precipitation, the 319 amino acid protein was purified by column chromatography on phenyl-Sepharose and heparin-Sepharose. Annexin V elutes on reverse phase C4 silica as a single peak with greater than 97% homogeneity and is further characterized by a molecular mass of 34 kDa from electrophoresis under reducing conditions on SDS gels. Dynamic light scattering experiments reveal annexin V exists as a monomer in solution. Amino terminal Edman degradation afforded no sequence, therefore the carbamidomethylated protein was chemically cleaved with cyanogen bromide. Separation of the resulting peptide fragments on reverse phase HPLC followed by N-terminal sequencing and electrospray mass spectrometry supported the correct sequence as well as the existence of an acetyl blocking group on the N-terminus. The protein exhibits an isoelectric point of 4.73 by column chromatofocusing. Secondary structure predictions from CD spectroscopy indicate that the molecule is correctly folded. In anticoagulant assays, the purified protein exhibits dose-response effects in activated partial thromboplastin time (APTT) prolongation and doubles the clotting time of control human plasma at 70 micrograms ml-1. More specifically, in a factor Xa inhibition assay in which the activation of factor X via the tissue factor-factor VIIa complex is monitored by the cleavage of a factor Xa chromogenic substrate, recombinant annexin V exhibits a 50% inhibitory concentration (IC50) in the low nanomolar range.

Annexins are intracellular proteins which bind to membranes in a Ca(2+)-dependent manner and which have been proposed to play regulatory roles in different membrane processes. In the present study, the stoichiometry of the Ca(2+)-dependent binding of annexin V to phosphatidylserine molecules incorporated into liposomes was studied by fluorescence spectroscopy. The Ca(2+)-dependence of the binding was determined using liposomes made of dioleoylphosphatidylserine (PS) and dioleoylphosphatidylcholine (PC), with a PC/PS molar ratio ranging from 1 to 800. These liposomes were shown to be mostly unilamellar by cryoelectron microscopy. [Ca2+]1/2 concentrations required for half-maximal binding of annexin V range from 57 microM at PC/PS = 1 up to 96 mM at PC/PS = 800. Titration of accessible PS molecules showed that annexin V molecules bind equally well to liposomes of PC/PS ratio ranging from 1 to 400. The stoichiometry of the binding between annexin V and PS, determined at low PS content, is eight annexin V molecules per one PS molecule. We propose a novel model of the Ca(2+)-dependent interaction between annexin V and lipid membranes, based on the formation of two-dimensional arrays of annexin V molecules, stabilized by both protein-lipid and protein-protein interactions.

We isolated and purified 35 kDa protein from the myocardium of the beagle dog and identified it to be annexin V from partial amino acid sequence determination. It was confirmed that anticanine cardiac annexin V rabbit polyclonal antibody, which was produced using the 35 kDa protein, cross-reacts with annexin V of the myocardium, lung, liver, kidney, and brain of the rat. The localization of cardiac annexin V and the effect of ischemia for 30-180 min in the rat were immunohistochemically studied with the use of the Langendorff perfusion heart. In the normal myocardium, annexin V, accompanied by cross-striation, was observed throughout the cell. In ischemia of 30 min, extracellular leakage of annexin V was observed with uneven staining in the cytoplasm. When the ischemic time exceeded 60 min, annexin V was observed in the cell membrane with a decrease of annexin V in the cytoplasm. Also, extracellular leakage of annexin V was observed prominently. In ischemia for 180 min, almost all the annexin V in the cytoplasm disappeared. These results suggest that the level of ischemia can be estimated from the changes in localization of annexin V.

Binding of viral envelope proteins to specific receptors on human hepatocytes is considered to be an important step in HBV infection. In this study, we demonstrate that a 34-kDa human liver plasma membrane protein specifically binds to small HBsAg in a Ca(2+)-dependent manner. By partial amino acid sequence analysis of preparatively isolated 34-kDa protein comigrating with HBsAg-binding protein obtained from binding assay on IEF/SDS-PAGE, we have identified this HBsAg-binding protein as Endonexin II (E-II). Native human liver E-II inhibits binding of HBsAg to intact human hepatocytes and shows specific binding to small HBsAg. This binding can be inhibited by human liver plasma membrane proteins, recombinant E-II, or anti-E-II antibodies. Despite 90% sequence homology, rat liver E-II does not bind to small HBsAg and does not inhibit significantly (less than 20%) binding of HBsAg to intact hepatocytes. Cross-linking of small HBsAg and radiolabeled human liver E-II resulted in a specific additional protein complex on PAGE with an apparent molecular weight of 90 kDa, corresponding to a complex of E-II and small HBsAg with a ratio of 2 to 1 or 1 to 2. These findings indicate that E-II, found in human liver, is a specific HBsAg-binding protein and might play an important role in the initiation of HBV infection.

Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.

Objective: The heart contains proteins of the annexin family, a unique group of calcium binding proteins. This study was aimed at identifying the major cardiac annexins and determining their distribution in the rat heart., Methods: Annexins were isolated by affinity chromatography and purified by ion exchange high pressure liquid chromatography. Identification of isolated proteins by immunoblotting was confirmed by partial amino acid sequence determination. Antisera raised against the isolated proteins were used for immunohistochemistry by the avidin-biotin-peroxidase technique., Results: Two annexins were isolated and purified. Amino acid sequencing confirmed their identities as annexin V and VI. Immunohistochemistry showed that both annexins were present in cardiac myocytes and non-myocytes, but a distinct pattern of distribution was seen for each annexin. Annexin V immunoreactivity was enhanced in the atria compared with the ventricles, whereas annexin VI was more uniformly distributed. In individual cardiac myocytes annexin V was distributed throughout the cell by contrast with annexin VI, which localised to the sarcolemma. Intercalated discs displayed immunoreactivity for both annexins, most prominently for annexin VI. The most striking immunoreactivity for annexin V occurred in vascular endothelial cells, both in the microcirculation and in the major coronary vessels. Immunoreactivity for annexin VI in vascular structures was localised to the nuclei of endothelial and smooth muscle cells., Conclusions: Annexins V and VI are the major cardiac annexins. The localisation of these annexins to different components of cardiac myocytes will serve to direct the search for their functions in the heart. The striking immunoreactivity for annexins, particularly annexin V, in the coronary vessels indicates that the functions of cardiac annexins include a role in the coronary circulatory system.

In vitro phosphorylation of annexin 1 by purified rat brain protein kinase C (PKC) has been studied in the presence of annexin 5, which is not a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing the concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annexin 5 and the residual phosphorylation of annexin 1. These data fit with the 'surface depletion model' explaining the antiphospholipase activity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have been quantified in human endothelial cells by measuring the radioactivity bound to the proteins after Western blotting with specific antibodies and 125I-labelled secondary antibody. Our data indicate that annexins 1 and 5, PKC and PtdSer are present in human endothelial cells in relative amounts very similar to those used in vitro under conditions permitting the detection of the inhibitory effect of annexin 5. Since annexin 1 remained refractory to PKC-dependent phosphorylation in intact cells, we suggest that annexin 5 might exert its inhibitory effect towards PKC in vivo, provided that its binding to phospholipids can occur at physiological (micromolar) concentrations of Ca2+. This was previously shown to occur in vitro using phosphatidylethanolamine/phosphatidic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-200]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation without interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PKC. Whether a similar mechanism also occurs in vivo remains to be determined.

The crystal structure of chicken annexin V has been solved by molecular replacement and refined at 2.25 A. The final R factor is 19.7% with good geometry. The chicken annexin V structure is very similar to the human annexin V structure, with four similar domains each containing five helices. The structure includes three calcium ions in domains I, II, and IV, each bound by the characteristic K-G-X-G-T-(38 residues)-D/E motif. In view of the structural similarity between human and chicken annexin V, we suggest that they have a common vital function which developed early in evolutionary history.