The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex.

The tyrosine kinase substrate annexin II is a member of a multigene family of Ca2+ and lipid-binding proteins which have been implicated in a number of membrane-related events. We have analyzed the subcellular distribution of annexin II in relation to other cellular components in normal and specifically manipulated MDCK cells. In a polarized monolayer of MDCK cells annexin II and its cellular ligand p11 are restricted almost exclusively to the cortical regions of the cells which also contain peripheral early endosomes. Treatment of the polarized cells with low Ca2+ medium leads to a disintegration of the cortical cytoskeleton and a translocation of both, the annexin II2p11(2) complex and early endosomes, to the cytoplasm. A similar translocation which is however specific for the annexin II2p11(2) complex and early endosomes and does not affect other elements of the cell cortex is observed in cells expressing a trans-dominant annexin II-p11 mutant. This chimeric mutant protein causes the aggregation of endogenous annexin II and p11 and the simultaneous detachment of early endosomes from the cell periphery resulting in the binding of the early endosomes but no other components of the endocytotic or biosynthetic pathways to the annexin II/p11 aggregates. The specificity of this effect argues for the association of the annexin II2p11(2) complex with early endosomes and suggests that this association contributes to establish the peripheral localization of early endosomal structures.

Characterization of the tubulin-tyrosine ligase.

The sequence of tubulin-tyrosine ligase (TTL), the enzyme catalyzing the ATP-dependent posttranslational addition of a tyrosine to the carboxyterminal end of detyrosinated alpha-tubulin, has been determined. TTL from bovine and porcine brain was purified by immunoaffinity chromatography and extensively characterized by protein sequencing. Oligonucleotides derived from the protein sequence were synthesized and partial cDNA sequences were obtained using reversed transcribed brain mRNA in polymerase chain reactions. Polymerase chain reaction fragments were used to isolate a full-length cDNA clone from a randomly primed lambda gt10 cDNA library obtained from embryonic porcine brain mRNA. Porcine TTL is encoded by 1,137 nucleotides corresponding to 379 amino acid residues. It has a molecular weight of 43,425 and a calculated isoelectric point of 6.51. Northern blot analysis revealed a surprisingly long mRNA (approximately 6 kb in embryonic porcine brain). The protein sequence of TTL shares no extended homology with the sequences in the data banks. TTL contains a potential serine phosphorylation site for cAMP-dependent protein kinase (RKAS at positions 73 to 76). Residues 244 to 258 lie at the surface of the molecule. A rabbit antibody raised against a synthetic peptide corresponding to this sequence binds to native TTL. The same sequence contains the cleavage site for endoproteinase Glu-C (residue 248) previously shown to convert TTL into a nicked derivative in which the two fragments still form a tight complex but don't display enzymatic activity.

Annexin II is a major component of fusogenic endosomal vesicles.

We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two-dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.

Combined homology modelling and evolutionary significance evaluation of missense mutations in blood clotting factor VIII to highlight aspects of structure and function.

Most small lesions in the factor VIII (FVIII) gene that cause haemophilia A (HA) are single nucleotide substitutions resulting in amino acid replacing (missense) mutations and leading to various phenotypes, ranging from mild to severe. We took a combined approach of homology modelling and quantitative evaluation of evolutionary significance of amino acid replacing alterations using the Grantham Matrix Score (GMS) to assess their structural effects and significance of pathological expression. Comparative homology models of all amino acid substitutions summarized in the FVIII mutations database plus these identified and reported lately by us or by our collaborators were evaluated. Altogether 640 amino acid replacing mutations were scored for potential distant or local conformation changes, influence on the molecular stability and predicted contact residues, using available FVIII domain models. The average propensity to substitute amino acid residues by mutation was found comparable to the overall probability of de novo mutations. Missense changes reported with various HA phenotypes were all confirmed significant using GMS. The fraction of these, comprising residues apparently involved in intermolecular interactions, exceeds the average proportion of such residues for FVIII. Predicted contact residues changed through mutation were visualized on the surface of FVIII domains and their possible functional implications were verified from the literature and are discussed considering available structural information. Our predictive modelling adds on the current view of domain interface molecular contacts. This structural insight could aid in part to the design of engineered FVIII constructs for therapy, to possibly enhance their stability and prolong circulating lifetime.

Annexin 2 has an essential role in actin-based macropinocytic rocketing.

Annexin 2 is a Ca(2+) binding protein that binds to and aggregates secretory vesicles at physiological Ca(2+) levels [1] and that also associates Ca(2+) independently with early endosomes [2, 3]. These properties suggest roles in both exocytosis and endocytosis, but little is known of the dynamics of Annexin 2 distribution in live cells during these processes. We have used evanescent field microscopy to image Annexin 2-GFP in live, secreting rat basophilic leukemia cells and in cells performing pinocytosis. Although we found no evidence of Annexin 2 involvement in exocytosis, we observed an enrichment of Annexin 2-GFP in actin tails propeling macropinosomes. The association of Annexin 2-GFP with rocketing macropinosomes was specific because Annexin 2-GFP was absent from the actin tails of rocketing Listeria. This finding suggests that the association of Annexin 2 with macropinocytic rockets requires native pinosomal membrane. Annexin 2 is necessary for the formation of macropinocytic rockets since overexpression of a dominant-negative Annexin 2 construct abolished the formation of these structures. The same construct did not prevent the movement of Listeria in infected cells. These results show that recruitment of Annexin 2 to nascent macropinosome membranes 16656is an essential prerequisite for actin polymerization-dependent vesicle locomotion.

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.

Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells.

The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.

Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I.

BACKGROUND: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. RESULTS: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. CONCLUSIONS: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

CC chemokine receptor 10 cell surface presentation in melanocytes is regulated by the novel interaction partner S100A10.

The superfamily of G-protein-coupled receptors (GPCR) conveys signals in response to various endogenous and exogenous stimuli. Consequently, GPCRs are the most important drug targets. CCR10, the receptor for the chemokines CCL27/CTACK and CCL28/MEC, belongs to the chemokine receptor subfamily of GPCRs and is thought to function in immune responses and tumour progression. However, there is only limited information on the intracellular regulation of CCR10. We find that S100A10, a member of the S100 family of Ca(2+) binding proteins, binds directly to the C-terminal cytoplasmic tail of CCR10 and that this interaction regulates the CCR10 cell surface presentation. This identifies S100A10 as a novel interaction partner and regulator of CCR10 that might serve as a target for therapeutic intervention.

The annexin II2p11(2) complex is the major protein component of the triton X-100-insoluble low-density fraction prepared from MDCK cells in the presence of Ca2+.

Annexin II2p11(2) is present in the submembranous region of cells expressing both subunits of the complex. Most probably, this subcellular distribution is maintained through the interaction of annexin II2p11(2) with membrane phospholipids and/or elements of the cortical cytoskeleton known to occur in vitro in a Ca(2+)-dependent manner. To determine whether membrane or cytoskeleton interactions are primarily responsible for anchoring annexin II2p11(2) in the cell cortex, we subjected Madin-Darby canine kidney (MDCK) cells to serial extractions using different detergents and identified annexin II and p11 in the different fractions employing specific antibodies. The complex but not monomeric annexin II remains insoluble when the cells are extracted with Triton X-100 in the presence of Ca2+. However, treatment of the Triton X-100-insoluble cell remnants with a series of other detergents known to solubilize GPI-anchored proteins leads to a partial extraction of annexin II2p11(2) even in the presence of Ca2+. Using sucrose density gradient analysis in the presence of Ca2+ as a different means of fractionating the Triton X-100-insoluble cell remnants we show that the majority of annexin II2p11(2) copurifies with a low-density fraction which has been reported to contain GPI-anchored proteins, certain glycolipids, and VIP21/caveolin. Annexin II2p11(2) is by far the most abundant protein component in this fraction indicating that its association with the low-density material occurs via lipid binding and is not due to the interaction with a certain protein.

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins.

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.

Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II.

Annexin II is a Ca(2+)-regulated membrane- and cytoskeleton-binding protein implicated in membrane transport events along the Ca(2+)-regulated secretory and the early endocytic pathway. Biochemical properties of this annexin and its intracellular distribution are regulated by complex formation with p11 (S100A10), a member of the S100 protein family. The annexin II-p11 interaction is mediated through the unique N-terminal domain of annexin II and is inhibited by protein kinase C phosphorylation of a serine residue in annexin II. To map this regulatory serine phosphorylation site we developed a baculovirus-mediated expression system for wild-type annexin II and for a series of annexin II mutants which contained substitutions in one or more serine residues present in the N-terminal domain. The different mutant derivatives were purified and shown to display the same biochemical properties as recombinant wild-type annexin II and the authentic protein purified from porcine intestine. However, significant differences in phosphate incorporation were observed when the individual serine mutants were subjected to phosphorylation by protein kinase C. A comparison of the phosphorylation patterns obtained identified Ser-II as the protein kinase C site responsible for regulating the annexin II-p11 interaction. Ser-II lies within the sequence mediating p11 binding, i.e. amino-acid residues 1 to 14 of annexin II, and phosphorylation at this site most likely leads to a direct spatial interference with p11 binding.

Phospholipid vesicle binding and aggregation by four novel fish annexins are differently regulated by Ca2+.

Four members of the annexin family, herein referred to as max (for medaka annexin) 1-4, have recently been identified through hybridization cloning in the killifish Oryzias latipes (D. Osterloh, J. Wittbrodt and V. Gerke, Characterization and developmentally regulated expression of four annexins in the killifish medaka. DNA and Cell Biol., in press). These annexins which are expressed in a developmentally regulated manner are present as a maternal pool in unfertilized eggs of another fish species, Misgurnus fossilis, and it has been proposed that they play a role in the Ca2+-regulated exocytosis of cortical granules occurring after fertilization. To characterize biochemical properties of the medaka proteins possibly relevant to their function in early development, we analyzed the ability of recombinantly expressed max 1-4 to interact with the principal structures of the egg cortex, phospholipid membranes and actin filaments. We show that all medaka annexins bind to acidic phospholipids in a Ca2+-regulated manner, although exhibiting different Ca2+ sensitivities. All medaka annexins, but max 1, are also capable of inducing, in a Ca2+-dependent manner, phospholipid vesicle aggregation, albeit only max 3 displays this activity at Ca2+ concentrations met in stimulated (i.e. fertilized) eggs. Max 3 is also the only medaka annexin able to interact with F-actin in the presence of Ca2+. These data identify by biochemical criteria max 3 as a close relative of the mammalian annexins I and II, thus supporting previous sequence-based comparisons. Max 3 is therefore the prime annexin candidate for being involved in cortical granule exocytosis, possibly by providing granule granule, granule plasma membrane and/or granule cytoskeleton contacts.

Polyisoprenylation of the CAAX motif--an in vitro protein synthesis study.

A number of proteins, including most nuclear lamins, certain fungal mating pheromones, G-protein gamma-subunits and ras proteins, contain a C-terminal cysteine-aliphatic-aliphatic-undefined amino acid (CAAX) motif which is thought to be a roughly defined consensus sequence capable of directing a series of posttranslational events, beginning with the addition of a polyisoprene moiety to the cysteine. So far such a motif has been found in every protein known to have this type of modification. We have utilized the rabbit reticulocyte lysate translation system, which is capable of carrying out the polyisoprene modification in vitro, to investigate features of the C-terminal motif which affect its suitability as a substrate. We demonstrate that a cysteine is only isoprenylated when situated at position -4 from the C-terminus. We further show that the presence of a glycine at position -3 or a terminal aromatic residue, features typical of some G-protein alpha subunits, cause a reduction and abolition respectively of isoprenylation.

X-ray structure of full-length annexin 1 and implications for membrane aggregation.

Annexins comprise a multigene family of Ca2+ and phospholipid- binding proteins. They consist of a conserved C-terminal or core domain that confers Ca2+-dependent phospholipid binding and an N-terminal domain that is variable in sequence and length and responsible for the specific properties of each annexin. Crystal structures of various annexin core domains have revealed a high degree of similarity. From these and other studies it is evident that the core domain harbors the calcium-binding sites that interact with the phospholipid headgroups. However, no structure has been reported of an annexin with a complete N-terminal domain. We have now solved the crystal structure of such a full-length annexin, annexin 1. Annexin 1 is active in membrane aggregation and its refined 1.8 A structure shows an alpha-helical N-terminal domain connected to the core domain by a flexible linker. It is surprising that the two alpha-helices present in the N-terminal domain of 41 residues interact intimately with the core domain, with the amphipathic helix 2-12 of the N-terminal domain replacing helix D of repeat III of the core. In turn, helix D is unwound into a flap now partially covering the N-terminal helix. Implications for membrane aggregation will be discussed and a model of aggregation based on the structure will be presented.

Folding energetics of ligand binding proteins II. Cooperative binding of Ca2+ to annexin I.

The calcium binding properties of annexin I as observed by thermodynamic DSC studies have been compared to the structural information obtained from X-ray investigation. The calorimetric experiment permitted to evaluate both the reaction scheme - including binding of ligand and conformational changes - and the energetics of each reaction step. According to published X-ray data Annexin I has six calcium binding sites, three medium-affinity type II and three low-affinity type III sites. The present study shows that at 37 degrees C annexin I binds in a Hill type fashion simultaneously two calcium ions in a first step with medium affinity at a concentration of 0.6 mM and another three Ca(2+) ions again cooperatively at 30 mM with low affinity. Therefore it can be concluded that only two medium-affinity type II binding sites are available. The third site, that should be accessible in principle appears to be masked presumably due to the presence of the N terminus. In view of the large calcium concentration needed for saturation of the binding sites, annexin I may be expected to be Ca(2+) free in vivo unless other processes such as membrane interaction occur simultaneously. This assumption is consistent with the finding, that the affinity of annexins to calcium is usually markedly increased by the presence of lipids.

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy.

The (annexin II-p11)2 tetramer has been proposed to participate in exocytosis and several other members of the annexin superfamily have been reported to aggregate liposomes in vitro. In this context, the Ca2+-dependent binding of several annexins to chromaffin granules and liposomes was investigated by cryo-electron microscopy. The Ca2+-dependent aggregation of lipid membranes by (annexin II-p11)2 results from the spontaneous self-organization of the protein into two-dimensional plaques, which are visualized in projection as characteristic junctions. The junctions have a constant thickness of 210(+/-10) A and present a symmetrical distribution of electron-dense material arranged into seven stripes. They were observed over a wide range of Ca2+ concentrations, down to 2 microM. The molecular components corresponding to the seven electron-dense stripes were assigned as follows: the two associated membranes give rise to two outer stripes each and the three central stripes correspond to the (annexin II-p11)2 tetramer. Each annexin II molecule interacts with the outer lipid leaflet of one membrane, giving rise to one stripe, while the central stripe is due to the (p11)2 dimer with which both annexin II molecules interact. Both annexin II and annexin I also induced the Ca2+-dependent aggregation of liposomes via junctions that lack the central (p11)2 moiety and present only six high-density stripes. As expected, both annexin V and annexin III bind to liposomes without inducing their aggregation.

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.