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The S100A3 homotetramer assembles upon citrullination of a specific symmetric Arg51 pair on its homodimer interface in human hair cuticular cells. Each S100A3 subunit contains two EF-hand-type Ca(2+)-binding motifs and one (Cys)3His-type Zn(2+)-binding site in the C-terminus. The C-terminal coiled domain is cross-linked to the presumed docking surface of the dimeric S100A3 via a disulfide bridge. The aim of this study was to determine the structural and functional role of the C-terminal Zn(2+)-binding domain, which is unique to S100A3, in homotetramer assembly. The binding of either Ca(2+) or Zn(2+) reduced the α-helix content of S100A3 and modulated its affinity for the other cation. The binding of a single Zn(2+) accelerated the Ca(2+)-dependent tetramerization of S100A3 while inducing an extensive unfolding of helix IV. The Ca(2+) and Zn(2+) binding affinities of S100A3 were enhanced when the other cation bound in concert with the tetramerization of S100A3. Small angle scattering analyses revealed that the overall structure of the S100A3 tetramer bound both Ca(2+) and Zn(2+) had a similar molecular shape to the Ca(2+)-bound form in solution. The binding states of the Ca(2+) or Zn(2+) to each S100A3 subunit within a homotetramer appear to be propagated by sensing the repositioning of helix III and the rearrangement of the C-terminal tail domain. This article is part of a Special Issue entitled: 12th European Symposium on Calcium., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The Receptor for Advanced Glycation Endproducts (RAGE) is a multiligand receptor involved in a large number of human disorders. Identified first as the receptor for the Advanced Glycation Endproducts (AGEs), RAGE has emerged in recent years as a major receptor for many members of the S100 calcium and zinc binding protein family. The interaction with and the signaling triggered by several S100 proteins such as S100B and S100A12 have been studied in details and have shown concentration and cell type dependent signaling cascades. The S100 protein family consists of more than 20 members which present high amino-acid sequence and structural similarities. These small EF-hand calcium binding proteins interact with a large number of protein targets and are almost all been shown to be involved in cancer. In this review we discuss the recent knowledge about the role of S100 proteins and RAGE in human disorders.

Mitochondrial Ca(2+) signals have been proposed to accelerate oxidative metabolism and ATP production to match Ca(2+)-activated energy-consuming processes. Efforts to understand the signaling role of mitochondrial Ca(2+) have been hampered by the inability to manipulate matrix Ca(2+) without directly altering cytosolic Ca(2+). We were able to selectively buffer mitochondrial Ca(2+) rises by targeting the Ca(2+)-binding protein S100G to the matrix. We find that matrix Ca(2+) controls signal-dependent NAD(P)H formation, respiration, and ATP changes in intact cells. Furthermore, we demonstrate that matrix Ca(2+) increases are necessary for the amplification of sustained glucose-dependent insulin secretion in β cells. Through the regulation of NAD(P)H in adrenal glomerulosa cells, matrix Ca(2+) also acts as a positive signal in reductive biosynthesis, which stimulates aldosterone secretion. Our dissection of cytosolic and mitochondrial Ca(2+) signals reveals the physiological importance of matrix Ca(2+) in energy metabolism required for signal-dependent hormone secretion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

S100B is a homodimeric zinc-, copper-, and calcium-binding protein of the family of EF-hand S100 proteins. Zn(2+) binding to S100B increases its affinity towards Ca(2+) as well as towards target peptides and proteins. Cu(2+) and Zn(2+) bind presumably to the same site in S100B. We determined the structures of human Zn(2+)- and Ca(2+)-loaded S100B at pH 6.5, pH 9, and pH 10 by X-ray crystallography at 1.5, 1.4, and 1.65Å resolution, respectively. Two Zn(2+) ions are coordinated tetrahedrally at the dimer interface by His and Glu residues from both subunits. The crystal structures revealed that ligand swapping occurs for one of the four ligands in the Zn(2+)-binding sites. Whereas at pH 9, the Zn(2+) ions are coordinated by His15, His25, His 85', and His 90', at pH 6.5 and pH 10, His90' is replaced by Glu89'. The results document that the Zn(2+)-binding sites are flexible to accommodate other metal ions such as Cu(2+). Moreover, we characterized the structural changes upon Zn(2+) binding, which might lead to increased affinity towards Ca(2+) as well as towards target proteins. We observed that in Zn(2+)-Ca(2+)-loaded S100B the C-termini of helix IV adopt a distinct conformation. Zn(2+) binding induces a repositioning of residues Phe87 and Phe88, which are involved in target protein binding. This article is part of a Special Issue entitled: 11th European Symposium on Calcium., (2010. Published by Elsevier B.V.)

Hallmarks of Alzheimer's disease (AD) include the accumulation of amyloid beta peptide (Abeta), hyperphosphorylation of tau protein, and increased inflammatory activity in the hippocampus and cerebral cortex. The receptor for advanced glycation endproducts (RAGE) has been shown to interact with Abeta and to modulate Abeta transport across the blood-brain barrier. Furthermore, RAGE is upregulated at sites of inflammation and its activation results in distinct intracellular signaling cascades in respect to Abeta conformers. Besides Abeta, RAGE interacts with several members of the calcium binding S100 protein family, amphoterin and advanced glycation endproducts. Mounting evidence suggests that RAGE is a key player in the signaling pathways triggered by Abeta and S100 proteins in AD. In this review, we discuss recent discoveries about the crosstalk between RAGE, Abeta and S100 proteins in the pathophysiology of AD.

The receptor for advanced glycation endproducts (RAGE) interacts with several ligands and is involved in various human diseases. RAGE_v1 or sRAGE, a RAGE splice variant, is secreted and contributes to the removal of RAGE ligands. Because RAGE blockade by specific antibodies directed against RAGE extracellular domains and the use of sRAGE have been proven to be beneficial in the context of pathological settings, both RAGE and sRAGE are considered as therapeutic target. Here, we show that sRAGE is also produced through regulated intramembrane proteolysis of the RAGE receptor, which is catalyzed by ADAM10 and the gamma-secretase and that calcium is an essential regulator of RAGE processing. Furthermore, RAGE intracellular domain localizes both in the cytoplasm and the nucleus and induces apoptosis when expressed in cells. These findings reveal new aspects of RAGE regulation and signaling and also provide a new interaction between RAGE and human pathologies.

Ca(2+) acts as global second messenger involved in the regulation of all aspects of cell function. A multitude of Ca(2+)-sensor proteins containing the specific Ca(2+) binding motif (helix-loop-helix, called EF-hand) developed early in evolution. Calmodulin (CAM) as the prototypical Ca(2+)-sensor with four EF-hands and its family members troponin-C (TNC), myosin light chains, and parvalbumin originated by gene duplications and fusions from a CAM precursor protein in prokaryotes. Rapid and precise regulation of heart and skeletal muscle contraction is assured by integration of TNC in the contractile structure and CAM in the sarcolemmal L-type Ca(2+) entry channel and in the sarcoplasmic Ca(2+) release channel RYR. The S100 proteins as evolutionary latecomers occur only in the animal subphylum vertebrates. They are not involved in switching on and off key cell functions but rather operate as modulators. In the heart S100A1 modulates Ca(2+) homeostasis, contractile inotropy, and energy production by interaction with the elements involved in these functions. The binding properties of different Ca(2+)-sensor proteins associated with specific regulatory and modulatory functions in muscle are discussed in detail. Some of these sensor proteins are critically involved in certain diseases and are now used in clinical diagnostics.

S100B, a Ca2+-binding protein, acts intracellularly as a Ca2+-signalling protein but is also secreted to the extracellular space, acting in a cytokine-like manner through its receptor RAGE. Recombinant human S100B has been purified and crystallized in the Ca2+-bound state. Size-exclusion chromatography indicates that S100B can exist as a dimer and as a multimer in solution. Crystals of S100B diffract to 1.9 A and belong to space group P2(1), with unit-cell parameters a = 63.4, b = 81.6, c = 71.5 A, alpha = 90, beta = 107, gamma = 90 degrees. Preliminary analysis of the X-ray data indicate that there are four homodimers per asymmetric unit.

NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, which is a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains (NDR(62-87)). Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis showed that the binding mode and stoichiometry of a peptide fragment of NDR (NDR(62-87)) is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B.

S100 proteins have attracted great interest in recent years because of their cell- and tissue-specific expression and association with various human pathologies. Most S100 proteins are small acidic proteins with calcium-binding domains - the EF hands. It is thought that this group of proteins carry out their cellular functions by interacting with specific target proteins, an interaction that is mainly dependent on exposure of hydrophobic patches, which result from calcium binding. S100A13, one of the most recently identified members of the S100 family, is expressed in various tissues. Interestingly, hydrophobic exposure was not observed upon calcium binding to S100A13 even though the dimeric form displays two high- and two low- affinity sites for calcium. Here, we followed the translocation of S100A13 in response to an increase in intracellular calcium levels, as protein translocation has been implicated in assembly of signaling complexes and signaling cascades, and several other S100 proteins are involved in such events. Translocation of S100A13 was observed in endothelial cells in response to angiotensin II, and the process was dependent on the classic Golgi-ER pathway. By contrast, S100A6 translocation was found to be distinct and dependent on actin-stress fibers. These experiments suggest that different S100 proteins utilize distinct translocation pathways, which might lead them to certain subcellular compartments in order to perform their physiological tasks in the same cellular environment.

In recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca(2+)- (Zn(2+)- and Cu(2+))-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca(2+) levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), the Ca(2+)-chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn(2+) levels. Treatment of cells with TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn(2+)-binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca(2+) levels, and even more so, a decrease in Zn(2+) concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway.

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.

The localization of S100A1 in rat soleus (SOL) and extensor digitorum longus (EDL) muscles was studied immunocytochemically at different sarcomere lengths (stretched, relaxed and contracted) at the ultrastructural level. The muscle fibres were contracted by application of 15 mmol/l caffeine. Following aldehyde fixation, dehydration and embedding in Lowicryl HM20 (-35 degrees C) ultrathin sections were incubated with rabbit polyclonal antiserum against S100A1. Goat antirabbit secondary antibodies conjugated with 10 nm gold particles were used to visualize antigen sites. Relative areas of Z-lines, A- and I-bands were estimated from longitudinal sections by the point counting method. The highest densities of the particles were found at the Z-lines. A higher incidence of S100A1 antigen sites in I-bands than in A-bands and a higher density of S100A1 in lateral parts of A-bands (with actin and myosin filaments overlapping) compared with the central area of A-bands are consistent with an interaction of S100A1 with F-actin in skeletal muscles. Antigen sites were also present at M-lines and at distinct locations of the sarcoplasmic reticulum.

The Ca(2+) binding protein S100A1 increases the Ca(2+) release from the sarcoplasmatic reticulum by interacting with the ryanodine receptor. In order to understand whether this effect might be operative in the early course of hypertrophy, when myocardium is able to meet increased workload, we investigated the expression of S100A1 in a model of moderate right ventricular hypertrophy. The pulmonary arteries of nine pigs were embolised three times with Sephadex G-50. After 70 days, all pigs showed a moderate pulmonary hypertension. Right ventricular tissue of embolised animals showed a significant increase of connective tissue and enlargement of myocyte diameters. In controls, we found a differential expression of S100A1 with significantly lower S100A1 protein levels in right ventricular compared to left ventricular tissue. In pulmonary hypertension, S100A1 expression increased significantly in hypertrophied right ventricles while it was unchanged in left ventricular tissue. No change was observed in the expression of SERCA2a and phospholamban. Our data show, for the first time, that moderate pressure overload results in an upregulation of S100A1. This may reflect an adaptive response of myocardial Ca(2+) homeostasis to a higher workload.

S100 proteins, a subgroup of the EF-hand Ca2+-binding protein family, regulate a variety of cellular processes via interaction with different target proteins. Several pathological disorders, including cancer, are linked to altered Ca2+ homeostasis and might involve the multifunctional S100 proteins, which are expressed in a cell- and tissue-specific manner. The present work demonstrates a distinct intracellular localization of S100A6, S100A4, and S100A2 in two tumor cell lines derived from metastatic epithelial breast adenocarcinoma (MDA-MB231) and cervical carcinoma (HeLa). Treatment of the cells by thapsigargin, the ionophore A23187, or cyclic ADP-ribose, to increase [Ca2+]i via different pathways, led to relocation of S100A6 and S100A4 but only partially of the nuclear S100A2, as demonstrated by confocal laser scanning microscopy. These findings support the hypothesis that S100 proteins could play a crucial role in the regulation of Ca2+ homeostasis in cancer cells.

A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca2+-binding proteins and other metalloproteins., (Copyright 1999 Academic Press.)

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.

Changes in cytosolic Ca2+ concentration control a wide range of cellular responses, and intracellular Ca2+-binding proteins are the key molecules to transduce Ca2+ signaling via interactions with different types of target proteins. Among these, S100 Ca2+-binding proteins, characterized by a common structural motif, the EF-hand, have recently attracted major interest due to their cell- and tissue-specific expression pattern and involvement in various pathological processes. The aim of our study was to identify the subcellular localization of S100 proteins in vascular smooth muscle cell lines derived from human aorta and intestinal smooth muscles, and in primary cell cultures derived from arterial smooth muscle tissue under normal conditions and after stimulation of the intracellular Ca2+ concentration. Confocal laser scanning microscopy was used with a specially designed colocalization software. Distinct intracellular localization of S100 proteins was observed: S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found predominantly in the cytosol where they were strongly associated with the sarcoplasmic reticulum and with actin stress fibers. In contrast, S100A2 was located primarily in the cell nucleus. Using a sedimentation assay and subsequent electron microscopy after negative staining, we demonstrated that S100A1 directly interacts with filamentous actin in a Ca2+-dependent manner. After thapsigargin (1 microM) induced increase of the intracellular Ca2+ concentration, specific vesicular structures in the sarcoplasmic reticulum region of the cell were formed with high S100 protein content. In conclusion, we demonstrated a distinct subcellular localization pattern of S100 proteins and their interaction with actin filaments and the sarcoplasmic reticulum in human smooth muscle cells. The specific translocation of S100 proteins after intracellular Ca2+ increase supports the hypothesis that S100 proteins exert several important functions in the regulation of Ca2+ homeostasis in smooth muscle cells.

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.

Human recombinant alpha-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca(2+)-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PVD51A,E62V,D90A,E101V), were expressed and purified. Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K'Ca, of 2.3 x 10(7) M-1 and that Mg2+ competes with a K'Mg.compet. of 4.9 x 10(3) M-1. The three mutants with an inactivated CD site bind 1 Ca2+ with K'Ca, of 2.0 to 2.3 x 10(7) M-1 and K'Mg.compet. of 3.4 to 4.6 x 10(3) M-1, i.e. very similar to those of PVwt. The mutants with an inactivated EF site bind 1 Ca2+ with K'Ca values of 7.9 x 10(6), 4.5 x 10(6) and 3.6 x 10(6) M-1 for PVD91A, PVE102V and PVE101V,D91A, respectively. The K'Mg.compet values of these mutants are about 4-times lower than in PVwt. The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+. After excitation at 259 nm, human PV, which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+. Specific antibodies against human alpha-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.

We investigated whether the expression of human alpha-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca2+]i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM. Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K+. During this period, [Ca2+]i was monitored by video microfluorimetry using the Ca2+ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca2+]i. The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca2+]i than non-parvalbumin-expressing cells. Resting [Ca2+]i did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca2+]i in neuroblastoma cells can be suppressed by parvalbumin.

Calcium ions regulate many processes in the central nervous system via interaction with intracellular calcium-binding proteins. One class of these proteins shares a common structural motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of many new members of this family. Some of these proteins, like parvalbumin, calbindin, and calretinin, proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major role is assumed to be buffering, transport of Ca2+, and regulation of various enzyme systems. Cellular degeneration is often accompanied by Ca2+ overload. It has been assumed that neurons containing certain intracellular Ca(2+)-binding proteins may have a greater capacity to buffer Ca2+ and therefore would be more resistant to degeneration.

The cellular distribution of Ca(2+)-binding proteins has been extensively studied during the past decade. These proteins have proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major roles are assumed to be Ca2+ buffering and transport, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca2+ homeostasis, a protective role for Ca(2+)-binding proteins in certain neuron populations has been postulated. As massive neuronal degeneration takes place in several brain diseases of humans, such as Alzheimer's disease, Parkinson's disease and epilepsy, changes in the expression of Ca(2+)-binding proteins have therefore been studied during the course of these diseases. Although the data from these studies are inconsistent, the detection and quantification of Ca(2+)-binding proteins and the neuron populations in which they occur may nevertheless be useful to estimate, for example, the location and extent of brain damage in the various neurological disorders. If future studies advance our knowledge about the physiological functions of these proteins, the neuronal systems in which they are expressed may become important therapeutical targets for preventing neuronal death in an array of neurodegenerative diseases.

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.

Under Ca2(+)-loaded conditions parvalbumin migrates in one- and two-dimensional gel-systems as a double-band or -spot whereas in Ca2(+)-free condition it appears as one band or spot. Parvalbumin (PV), a member of the family of calcium-binding proteins [1], was first described in 1934 [2] and occurs in fast-contracting muscles and in subpopulations of neurons in vertebrates and humans [3,4,5]. The physical characteristics of molecular weight (Mv 12 KD), isoelectric point (pI 4.9) and Ca2(+)-binding properties are established (PV binds 2 Ca2+ per molecule) [5,6]. Physiological roles discussed for PV range from trigger- to buffer-protein of intracellular Ca2(+)-ions [7]. In this paper we report an as yet not described mobility shift of PV in gel-electrophoresis after manipulation of the Ca2+ concentration, which may have implications for its physiological function.

Monoclonal antibody 235 which was used for immunohistochemical staining of parvalbumin in tissue sections partially protects Lys-54 of carp muscle parvalbumin from reaction with acetic anhydride in the parvalbumin-antibody complex. Lys-54 is located in the CD-loop of parvalbumin and is flanked by the Ca2(+)-ligands Asp-53 and Ser-55 of the Ca2(+)-site I. Another monoclonal antibody against carp parvalbumin, mca 239, partially protects lysine residues 27, 32, 87 and 107, indicating that this antibody is directed against a discontinuous epitope distant from the two Ca2(+)-binding sites of parvalbumin.

The Ca2+ pump in rat duodenal epithelium is studied as ATP-dependent Ca2+ uptake in a vesicle preparation with a 9-fold purification in Na+-K+-ATPase activity and a 20-fold purification of Na+-K+-ATPase with respect to an endoplasmic reticulum marker. ATP-dependent Ca2+ uptake is reduced by 60% by digitonin treatment of the vesicles, whereas high-affinity Ca2+-ATPase is stimulated by the same treatment. Different methods to deplete membrane preparations of calmodulin have been used. In EDTA osmotically shocked vesicles, calmodulin stimulated ATP-dependent Ca2+ transport up to 100% in a Ca2+ concentration-dependent manner. The duodenal Ca2+ pump is inhibited by calmodulin antagonists only at low Ca2+ concentrations and in membranes not depleted from calmodulin. Vitamin D-dependent Ca2+-binding protein (Mr = 10,000) in concentrations up to 5 microM did not affect the rate of ATP-dependent Ca2+ transport, either in Ca2+-EGTA-buffered solutions or in EGTA-free solutions. In membrane preparations from vitamin D-deficient rats, the effects of calmodulin and of Ca2+-binding protein were identical to the vitamin D-repleted control preparations. This excludes a specific effect of Ca2+-binding protein and calmodulin in the vitamin D dependency of duodenal Ca2+-ATPase.

Parvalbumin, a Ca2+-binding protein, was isolated from rat testis. This is the first demonstration of the protein in endocrine glands. By using a rat parvalbumin cDNA probe, parvalbumin mRNA was demonstrated in the testis, indicating that the protein is synthesized in this tissue and that testis parvalbumin is a product of the same gene as the one encoding for muscle parvalbumin. Parvalbumin was localized by immunohistochemical methods in the Leydig cells and in the acrosome region of maturing spermatids (stages 1-15). The expression of parvalbumin during testis development was followed. High parvalbumin protein and mRNA levels were found at stages of highest Leydig cell activity, i.e. at late fetal stages until birth and again around postnatal day 50. This suggests that parvalbumin may be involved in the production of testosterone in Leydig cells, a process which is highly dependent on calcium.

Calcium and intracellular Ca2+-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.

The fast extensor digitorum longus (EDL) and the slow soleus (SOL) muscles were cross-reinnervated in both directions in the rat. During the following transformation of muscle type properties, the expression of the Ca2+-binding parvalbumin (parvalbumin, Mr = 12,000) was investigated. The combined biochemical, histochemical, and physiological results demonstrated that the amount of parvalbumin decreased in the fast to slow (X-EDL) and increased in the slow to fast (X-SOL) transformation. Alterations of parvalbumin-mRNA levels were similar to changes found at the protein level, indicating a tight transcriptional regulation of the parvalbumin expression. The close correlation, however, between parvalbumin and relaxation speed found in normal muscles had changed after cross-reinnervation. After the altered nervous input, a slow contracting/slow relaxing muscle may even contain more parvalbumin than a fast contracting/fast relaxing one. The expression of parvalbumin may depend on the nerve-muscle interaction, and parvalbumin may thus be used as a sensitive marker for early stages of muscular transformation and neurological disorders.

The physiological role of the Ca2+-binding protein parvalbumin in skeletal muscle has been investigated by measuring the parvalbumin content by HPLC in a variety of mammalian muscles, including man, and comparing the results with the respective muscle relaxation properties and fiber type compositions. The parvalbumin concentrations were highest in the skeletal muscles of the smallest animal investigated (mouse, gastrocnemius: 4.9 g/kg), which has the highest relaxation speed, and lowest in the larger animals (horse, deep gluteal muscle: less than or equal to 0.001 g/kg) and man (vastus, triceps: less than or equal to 0.001 g/kg), which have much lower relaxation speeds. Analysis of three type-homogeneous muscles of the guinea pig revealed highest parvalbumin concentrations (0.25 g/kg) in sartorius (type IIB) and lowest concentrations (less than or equal to 0.007 g/kg) in soleus (type I), consistent with the different half-relaxation times of fast and slow muscles. Denervation of the rat extensor digitorum longus, which increases the half-relaxation time from 9.4 to 19 msec, resulted in a 20% decrease of the parvalbumin content. Given this close correlation between parvalbumin content and relaxation speed in a variety of muscles and species, we suggest that parvalbumin is involved directly in the relaxation process in fast muscles.