Annexin V, annexin VI, S100A1 and S100B in developing and adult avian skeletal muscles.

Annexins and S100 proteins constitute two multigenic families of Ca2+-modulated proteins that have been implicated in the regulation of both intracellular and extracellular activities. Some annexins can interact with certain S100 protein dimers thereby forming heterotetramers in which an S100 dimer crosslinks two copies of the partner annexin. It is suggested that S100 protein binding to an annexin might serve the function of regulating annexin function and annexin binding to an S100 protein might regulate S100 function. In the present study, annexin V, annexin VI (or ANXA5 and ANXA6, respectively, according to a novel nomenclature), S100A1 and S100B were analyzed for their subcellular localization in developing and adult avian skeletal muscles by confocal laser scanning microscopy, immunogold cytochemistry, and western blotting, and for their ability to form annexin-S100 heterocomplex in vivo by immunoprecipitation. These four proteins displayed distinct expression patterns, ANXA5 being the first to be expressed in myotubes (i.e. at embryonic day 8), followed by ANXA6 (at embryonic day 12) and S100A1 and S100B (between embryonic day 12 and embryonic day 15). The two annexins and the two S100 proteins were found associated to different extents with the sarcolemma, membranes of the sarcoplasmic reticulum, and putative transverse tubules where they appeared to be co-localized from embryonic day 18 onwards. No one of these proteins was found associated with the contractile apparatus of the sarcomeres. Immunoprecipitation studies indicated that ANXA6/S100A1 and ANXA6/S100B complexes formed in vivo. Whereas, ANXA5 was not recovered in S100A1 or S100B immunoprecipitates. From our data we suggest that: (i) ANXA5 and ANXA6, and S100A1 and S100B can be used as markers of skeletal muscle development; (ii) ANXA6 and S100A1 and S100B appear strategically located close to or on skeletal muscle membrane organelles that are critically involved in the regulation of Ca2+ fluxes, thus supporting previous in vitro observations implicating S100A1 and ANXA6 in the stimulation of Ca2+-induced Ca2+ release; and (iii) ANXA6/S100A1 and ANXA6/S100B complexes can form in vivo thereby regulating each other activities and/or acting in concert to regulate membrane-associated activities.

Ultracytochemical detection of guanylate cyclase C activity in alimentary tract and associated glands of the rat. Influence of pH, ATP and the ions Mg2+ and Mn2+.

Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg2+ and Mn2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg2+ or Mn2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum-ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg2+ and ATP at pH 8 and 7.4.

The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments.

The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix) type, S100A1 and S100B, that have been shown to inhibit microtubule (MT) protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF) subunits, desmin and glial fibrillary acidic protein (GFAP), with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 microM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head) domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B) represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

Ultracytochemical study of guanylate cyclases A and B in light- and dark-adapted retinas.

The ultracytochemical localization of guanylate cyclases A and B activity has been studied after stimulation with atrial natriuretic peptide and C-type natriuretic peptide in light- and dark-adapted retinas and pigmented epithelium. The results showed that both peptides stimulated guanylate cyclases A and B activity in light-adapted retinas only. Guanylate cyclases A and B activity was detected on plasma membrane of body of photoreceptors, bipolar, horizontal and ganglion cells, on plasma membranes of interneuronal connections at plexiform layers and on the plasma membrane of fibres at the nerve fibres layer. Independently of the light-or dark-adapted state, the pigmented epithelium also presented guanylate cyclases A and B activity on basal and lateral plasma membranes.

S100B and S100A1 proteins in bovine retina:their calcium-dependent stimulation of a membrane-bound guanylate cyclase activity as investigated by ultracytochemistry.

The Ca2(+)-binding proteins of the EF-hand type, S100B and S100A1, were detected in the outer segment of bovine retina photoreceptors where they are localized to disc membranes, as investigated by immunofluorescence and immunogold cytochemistry. S100B and S100A1 stimulate a membrane-bound guanylate cyclase activity associated with photoreceptor disc membranes in dark-adapted retina in a Ca2(+)-dependent manner, although with different Ca2+ requirements, as investigated by an ultracytochemical approach. Other retinal cell types express S100B and S100A1 as well. S100B is detected in the outer limiting membrane, fine cell processes in the outer nuclear layer and the outer plexiform layer, cell bodies in the inner nuclear layer and the ganglion cell layer, and the inner limiting membrane, whereas S100A1 has a more discrete distribution. S100B and S100A1 also stimulate a membrane-bound guanylate cyclase activity in photoreceptor cell bodies and Muller cells, but their effect appears independent of the light- or dark-adapted state of the retina and is observed at relatively high Ca2+ concentrations. These data represent the ultrastructural counterpart of recent biochemical observations implicating S100B and, possibly, S100A1 in the Ca2(+)-dependent stimulation of a photoreceptor membrane-bound guanylate cyclase activity [T. Duda, R. M. Goraczniak and R. K. Sharma (1996) Molecular characterization of S100A1-S1000B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclast. Biochemistry 35, 6263-6266; A. Margulis, N. Pozdnyakov and A. Sitaramayya (1996) Activation of bovine photoreceptor guanylate cyclast by S100 proteins. Biochem. Biophys. Res. Commun. 218, 243-247]. Our data suggest that at least S100B may take part in the regulation of a membrane-bound guanylate cyclase-based signalling pathway in both photoreceptors and Muller cells.

Replicating myoblasts and fused myotubes express the calcium-regulated proteins S100A1 and S100B.

We investigated the expression and the subcellular localization of S100A1 and S100B, two Ca(2+)-binding proteins of the EF-hand type, in replicating myoblasts and fused myotubes. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed the presence of S100A1 mRNA and S100B mRNA respectively, in myoblasts. Immunofluorescence and immunogold electron microscopy were used to localize individual proteins in myoblasts and myotubes. In the present report we document that: (1) in replicating myoblasts S100B is localized to intracellular membranes, including Golgi membranes, vimentin intermediate filaments (IFs) and microtubule (MT) structures; (2) in the same cells S100A1 is found associated with intracellular membranes; (3) following treatment of replicating myoblasts with colchicine, a fraction of S100B remains colocalized with bundled and collapsed vimentin IFs, whereas another fraction follows the destiny of endoplasmic membranes; (4) under the same conditions S100A1, like a fraction of S100B, follows the collapse of the endoplasmic reticulum around the nucleus; and (5) in fused myotubes S100A1 is found diffusely in the cytoplasm, whereas S100B is mostly found associated with vimentin IFs. These data suggest that in the skeletal myogenic cell line used in the present study S100A1 and S100B might share binding sites on or close to intracellular membranes, but display a significant degree of target specificity with respect of IFs and MTs. The results of these analyses suggest that expression of S100B in skeletal muscle cells may be developmentally regulated and lend support to the possibility that S100B might regulate the MT and IF dynamics.

Detection of guanylate cyclases A and B stimulated by natriuretic peptides in gastrointestinal tract of rat.

The ultracytochemical localization of membrane-bound guanylate cyclases A and B has been studied after stimulation with atrial natriuretic peptide, C-type natriuretic peptide and brain natriuretic peptide in the gastrointestinal tract of rat. The two isoforms are stimulated differently by the three peptides. The results showed that the atrial and C-type natriuretic peptides stimulated guanylate cyclase activity, whereas the brain peptide seemed not to activate enough of the enzyme to detect. The guanylate cyclase activity had a wider distribution in stomach and small intestine than in large intestine; nevertheless, the reaction product of guanylate cyclase A activity had a wider localization in the stomach, whereas the reaction product of guanylate cyclase B activity had a wider distribution in the small intestine. In the small and large intestine, we detected mostly similar localizations of guanylate cyclase activity irrespective of the peptide used; in the stomach the reaction products of guanylate cyclase A and B were detected in different cell types or in different sites of the same cell. In all the gastrointestinal tract, guanylate cyclase activity was detected mainly in three types of cells: exocrine and endocrine cells; undifferentiated and mature epithelial cells; and smooth muscle cells. These localizations of guanylate cyclase activity suggest its role in regulating glandular secretion, cellular proliferation and muscular activity.

Time-resolved fluorescence of S-100a protein: effect of Ca2+, Mg2+ and unilamellar vesicles of egg phosphatidylcholine.

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca(2+)-binding protein S-100a both in the absence and in the presence of Ca2+ and/or Mg2+. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca2+, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg2+, the Ca2+ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca2+, this value was increased to 16.08 ns. In the presence of Mg2+, Ca+2 increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg2+ indicate some interaction between lipids and S-100, likely mediated by this ion.

S-100 (alpha and beta) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP.

Alignment of previously characterized S-100 (alpha and beta)-binding peptides (J. Biol. Chem. 270, 14651-14658) has enabled the identification of a putative S-100 target epitope within the head domain of glial fibrillary acidic protein (GFAP). The capacity of a known peptide inhibitor of S-100 protein (TRTK-12), homologous to this region, to perturb the interaction of S-100 (alpha and beta) and GFAP (J. Biol. Chem 268, 12669-12674) was investigated. Fluorescence spectrophotometry and chemical cross-linking analyses determined TRTK-12 to disrupt S-100:GFAP interaction in a dose- and Ca(2+_dependent manner. TRTK-12 also inhibited S-100's ability to block GFAP assembly and to mediate disassembly of preformed glial filaments. Each of these events was strictly dependent upon the presence of calcium and inhibitory peptide, maximal inhibition occurring at a concentration of TRTK-12 equivalent to the molar amount of S-100 monomer present. Together with our recent report demonstrating TRTK-12 also blocks the interaction of S-100 protein with the actin capping protein, CapZ, these results suggest TRTK-12 functions as a pleiotropic inhibitor of S-100 function. Availability of a functional inhibitor of S-100 will assist the further characterization of S-100 protein function in vitro and in vivo. Moreover, this report provides additional evidence supportive of a role for S-100 as a multi-faceted regulator of cytoskeletal integrity.

Effects of calcium-binding proteins (S-100a(o), S-100a, S-100b) on desmin assembly in vitro.

S-100a(o), the alpha alpha isoform of a subfamily of Ca(2+)-binding proteins of the EF-hand type expressed in cardiac and skeletal muscle cells, is reported to inhibit the assembly of the intermediate filament subunit desmin and to stimulate the disassembly of desmin intermediate filaments in the presence of micromolar levels of free Ca(2+). These effects are dose-dependent with respect to the S-100a(o) concentration and maximal at a desmin/S-100a(o) (dimer) molar ratio of approximately 2. Other members of the S-100 subfamily [S-100a (alpha beta) and S-100b (beta beta) and the unfractionated mixture of S-100a plus S-100b produce qualitatively similar effects on desmin assembly, with a potency that depends on the fraction of S-100alpha subunit (the most potent) or S-100beta subunit (the least potent) present in the S-100 isoforms tested. A binding stoichiometry of 2 mol of desmin/mol of S-100a(o) (dimer) and an affinity in the submicromolar range are calculated. The S-100beta subunit also interacts with desmin, but with a lower affinity compared with S-100alpha. By contrast, the S-100-like proteins calcyclin and p11 neither interact with desmin nor affect desmin assembly. The present data suggest that S-100a(o) might play a role in the regulation of the state of assembly of desmin intermediate filaments.

Axotomy induces a different modulation of both low-affinity nerve growth factor receptor and choline acetyltransferase between adult rat spinal and brainstem motoneurons.

Adult rat spinal and brainstem motoneurons re-express low-affinity nerve growth factor receptor (p75) after their axotomy. We have previously reported and quantified the time course of this reexpression in spinal motoneurons following several types of injuries of the sciatic nerve. Other studies reported the reexpression of p75 in axotomized brainstem motoneurons. Results of these previous studies differed regarding the type of the most effective triggering injury for p75 reexpression, the relative duration of this reexpression and the decrease of choline acetyltransferase (ChAT) immunoreactivity (-IR) following a permanent axotomy of spinal or brainstem motoneurons. These differences suggest that these two populations of motoneurons respond to axotomy with a different modulation of p75 and ChAT expression. The aim of the present study was to determine whether differential modulation exists. We have analyzed and quantified the presence of p75- and ChAT-IR motoneurons in the hypoglossal nucleus following the same types of injury and the same time course we previously used for sciatic motoneurons. The results show that a nerve crush is the most effective triggering injury for p75 and that it induces similar temporal patterns of p75 and ChAT expression for sciatic and hypoglossal motoneurons. In contrast, a cut injury of the sciatic and hypoglossal nerves resulted in distinct temporal courses of both p75 and ChAT expression between these two populations of motoneurons. In fact, a permanent axotomy of the hypoglossal motoneurons induced i) a much longer maintenance phase for p75 than in sciatic motoneurons and ii) a progressive loss of ChAT-IR with a successive return to normal values in contrast to the modest decrease in the sciatic motoneurons. This evidence indicates that spinal and brainstem motoneurons respond to a permanent axotomy with a different modulation of p75 and ChAT expression. Altogether, the present data and the reported evidence of a differential post-axotomy cell death support the hypothesis that these two populations of motoneurons undergo different dynamic changes after axotomy.

Annexin II2-p11(2) (calpactin I) stimulates the assembly of GFAP in a calcium- and pH-dependent manner.

Annexin II2-p11(2) (calpactin I) was tested as a potential regulator of GFAP assembly into glial filaments (GF), following the observation that it interacts with GFAP and cosediments with GF in a sedimentation assay. Under conditions where GFAP assembly is reduced, e.g., at pH values > 6.8, annexin II2-p11(2) stimulates GF formation in a Ca(2+)- and dose-dependent manner. Concomitantly, an ever larger fraction of annexin II2-p11(2) can be recovered in GF pellets as the pH is raised from 6.8 to 7.35. Monomeric annexin II also stimulates GFAP assembly, although with a smaller efficacy as compared to annexin II2-p11(2), but does not cosediment with GF to a large extent, whereas p11 neither cosediments with GF nor affects GFAP assembly. On the other hand, the in vitro reconstituted annexin II2-p11(2) heterotetramer mimics native annexin II2-p11(2), and perturbation of the integrity of annexin II2-p11(2) by a mild treatment with alpha-chymotrypsin results in the nearly complete abolition of the stimulatory effect of annexin II2-p11(2) on GFAP assembly. These data suggest that annexin II2-p11(2) might be involved in the regulation of the state of assembly of GF, possibly in concert with other proteins.

S-100 protein and annexin II2-p11(2) (calpactin I) act in concert to regulate the state of assembly of GFAP intermediate filaments.

S-100 protein and annexin II2-p11(2) were reported to inhibit and to stimulate the assembly of glial fibrillary acidic protein (GFAP), respectively, in a Ca(2+)-dependent manner. Here we show by a number of experimental approaches that S-100 protein contrasts all the effects of annexin II2-p11(2) on GFAP assembly and, conversely, that annexin II2-p11(2) contrasts the inhibitory effects of S-100 protein on GFAP assembly, in a dose-dependent manner in both cases. Altogether, these data suggest that two specific Ca2+ effectors, i.e., annexin II2-p11(2) and S-100 protein, might regulate the state of assembly of glial filaments in a concerted manner.

Mechanism of S100 protein-dependent inhibition of glial fibrillary acidic protein (GFAP) polymerization.

S100 protein, a subfamily of Ca(2+)-binding proteins of the EF-hand type, was recently shown to bind to and to inhibit the polymerization of the glial fibrillary acidic protein (GFAP), the intermediate filament component of astroglial cells, in the presence of micromolar levels of Ca2+ (J. Biol. Chem. 268, 12669-12674). By a sedimentation assay and viscometry we show here that S100 protein interferes with the very early steps of GFAP polymerization (nucleation) and with the GFAP polymer growth, thereby retarding the onset of GFAP assembly, reducing the rate and the extent of GFAP assembly, and increasing the critical concentration of GFAP assembly. Moreover, S100 protein disassembles preformed glial filaments. All the above effects can be explained by sequestration of soluble GFAP by S100 protein, as also indicated by the stoichiometry of S100 protein binding to GFAP and of S100 protein effects on GFAP assembly. Our data suggest that S100 protein might serve the function of avoiding excess GFAP polymerization and might participate in remodeling of glial filaments following elevation of the intracellular free Ca2+ concentration. Also, our data lend support to the notion that intermediate filaments are dynamic cytoskeleton structures that assemble and disassemble, and to the existence of cytoplasmic factors implicated in the regulation of the state of assembly of intermediate filaments.

Calpactin I binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments in a Ca(2+)-dependent manner: implications for concerted regulatory effects of calpactin I and S100 protein on glial filaments.

Calpactin I, a heterotetrameric, cytoskeletal protein complex composed of two copies of annexin II cross-linked by two copies of p11, an S100-like protein, binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments (GF) in a Ca(2+)-dependent manner, apparently without affecting GFAP polymerization under the present experimental conditions. Cosedimentation of calpactin I with GF, which occurs at micromolar free Ca2+ concentrations, is proportional to the concentrations of both calpactin I and GFAP and does not occur under conditions where GFAP assembly is maximally inhibited by, e.g., S100 protein. Annexin II also cosediments with GF and binds to GFAP, although to much smaller extents. Other annexins, such as annexins I, V, and VI, or p11 do not bind to either GF or GFAP. Calpactin I and S100 protein bind to different sites on GFAP, as investigated by fluorescence spectroscopy using acrylodan-labeled GFAP. Calpactin I and S100 protein might act, in the presence of Ca2+, in a concerted manner to determine the number and topography of GF in differentiating and/or mature glial cells.

Annexins V and VI in rat tissues during post-natal development: immunochemical measurements.

Annexins V and VI, two Ca(2+)-dependent phospholipid- and membrane-binding proteins, were immunochemically measured in a number of rat organs and tissues during post-natal development. Annexin V proved much more abundant than annexin VI irrespective of the organ and the post-natal period considered. In the brain, annexin V accumulated at a high rate from the end of the first post-natal week onward, whereas annexin VI was expressed in extremely low amounts irrespective of the period investigated. In contrast, the levels of both annexins in the heart were nearly constant, in the post-natal period investigated. In skeletal muscles, annexin V and VI levels were high around post-natal day 1 and decreased thereafter. A similar pattern was observed for annexin V in liver, whereas the amounts of annexin VI in this organ were at the limits of detectability. In the lung, annexin V accumulated almost linearly from birth to adulthood, whereas annexin VI was relatively high at birth, decreased to low levels by the end of the first post-natal week, and re-accumulated thereafter. Among the organs examined, the lung and heart proved the richest sources of annexins V and VI. Annexin V appears to be a useful marker of and to be implicated in brain, lung and skeletal muscle maturation.

S-100 protein, but not calmodulin, binds to the glial fibrillary acidic protein and inhibits its polymerization in a Ca(2+)-dependent manner.

S-100 protein, a Ca(2+)-binding protein of the EF-hand type, interacts with the glial fibrillary acidic protein (GFAP) in a Ca(2+)-dependent manner. The binding of S-100 protein to GFAP was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and cross-linking experiments using the bifunctional cross-linker, disuccinimidyl suberate. The binding affinity was observed to be in the nanomolar range with a stoichiometry of 2 mol of GFAP/mol of S-100 protein (dimer). S-100 protein was found to inhibit the polymerization of GFAP in a dose- and Ca(2+)-dependent manner, with a half-maximal effect at an S-100 protein/GFAP molar ratio of 0.2 and maximal effect at a molar ratio of 0.5. Identical results were obtained irrespective of whether the unfractionated bovine brain S-100 protein mixture (S-100a plus S-100b), S-100ao, S-100a, or S-100b was used. S-100 protein was observed to be maximally effective as an inhibitor of GFAP polymerization at approximately 3 microM free Ca2+. Calmodulin neither bound to GFAP nor inhibited its polymerization. Altogether, the present results suggest that S-100 protein might be involved in the regulation of the state of assembly of glial filaments by binding to and sequestering unpolymerized GFAP.

Immunocytochemical analyses of annexin V (CaBP33) in a human-derived glioma cell line. Expression of annexin V depends on cellular growth state.

The subcellular distribution of annexin V, a calcium-dependent phospholipid- and membrane-binding protein, in a human-derived cell line, GL15, was investigated by immunocytochemistry at light and electron microscope levels. Annexin V was found diffusely in the cytoplasm and associated with plasma membranes, membranes delimiting cytoplasmic vacuoles, membranes of the endoplasmic reticulum, and filamentous structures the identity of which remains to be established. By immunocytochemistry at the light microscope level and immunochemistry, the expression of annexin V in these cells was found to depend on cellular growth stage, being maximal soon after plating and progressively declining thereafter. However, re-expression of annexin V was observed whenever cell proliferation slowed down or arrested. These findings suggest that annexin V in glioma cells is mostly expressed in connection with cell differentiation. Also, the present ultrastructural data suggest that plasma membranes, membranes of the endoplasmic reticulum and the cytoskeleton are prominent sites of action of annexin V in vivo, thus lending support to the possibility that this protein might have a role in the regulation of cytoskeleton elements and/or of the structural organization of membranes.

Time-resolved fluorescence of S-100a protein in the absence and presence of calcium and phospholipids.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca(2+)-binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca2+. Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca2+. Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca2+. Our data suggest that the conformation of the protein and its Ca(2+)-binding properties vary depending on the characteristics of charge and structure of phospholipids.

S-100 protein binds to annexin II and p11, the heavy and light chains of calpactin I.

S-100 protein, a dimeric, Ca(2+)-binding protein of the EF-hand type, interacts with annexin II (p36, the heavy chain of the cytoskeletal protein complex, calpactin I), with p11 (the light and regulatory chain of calpactin I) and with the hetero-tetramer annexin II2-p11(2) (calpactin I) in a Ca(2+)-regulated way, but not with annexins I, V and VI. The interaction of S-100 protein with the above proteins was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and acrylodan-annexin II and by cross-linking experiments using the bifunctional cross-linker disuccinimidyl suberate (DSS). S-100 protein binds with the highest affinity to annexin II (Kd approx. 0.4 microM) and with the lowest affinity to calpactin I (Kd approx. 10 microM), with a constant stoichiometry of about 2 mol of protein/S-100 dimer. Thus, S-100 protein could substitute for p11 in regulating the activities of annexin II in cells which do not express p11 and/or act synergistically with p11 in cells expressing both p11 and S-100. The binding of S-100 protein to p11 could reflect the natural tendency of S-100 subunits and p11 to dimerize. Chimeric p11-S-100 alpha and p11-S-100-beta proteins could therefore form in a Ca(2+)-regulated way. The interaction of S-100 protein with calpactin I appears of doubtful physiological importance, because of the low binding affinity, of the small extent of fluorescence changes induced by calpactin I in acrylodan-S-100 protein and of lack of DSS-induced complex formation between the two protein species.