High Affinity Calcium-binding Protein Research Articles

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Loss of Doc2b does not influence transmission at Purkinje cell to deep nuclei synapses under physiological conditions.

Doc2a and Doc2b are high-affinity calcium-binding proteins that interact with SNARE proteins and phospholipids. Experiments performed on cultured cells indicated that Doc2 proteins promote spontaneous vesicle fusion and asynchronous neurotransmitter release, regulate vesicle priming, mediate augmentation, and regulate transmission during sustained activity. Here, we assess the role of Doc2 proteins in synaptic transmission under physiological conditions at mature synapses made by Purkinje cells onto neurons in the deep cerebellar nuclei (PC to DCN synapses). PCs express Doc2b but not Doc2a. Surprisingly, spontaneous neurotransmitter release, synaptic strength, the time course of evoked release, responses evoked by sustained high-frequency stimulation, and short-term plasticity were normal in Doc2b KO mice. Thus, in stark contrast to numerous functions previously proposed for Doc2, here we find that Doc2b removal does not influence transmission at PC-to-DCN synapses, indicating that conclusions based on studies of Doc2b in cultured cells do not necessarily generalize to mature synapses under physiological conditions.

Current Understanding of the Role of Neuronal Calcium Sensor 1 in Neurological Disorders.

Neuronal calcium sensor 1 (NCS-1) is a high-affinity calcium-binding protein and its ubiquitous expression in the nervous system implies a wide range of functions. To date, it has been implicated in regulation of calcium channels in both axonal growth cones and presynaptic terminals, pre- and postsynaptic plasticity mechanisms, learning and memory behaviors, dopaminergic signaling, and axonal regeneration. This review summarizes these functions and relates them to several diseases in which NCS-1 plays a role, such as schizophrenia and bipolar disorder, X-linked mental retardation and fragile X syndrome, and spinal cord injury. Many questions remain unanswered about the role of NCS-1 in these diseases, particularly as the genetic factors that control NCS-1 expression in both normal and diseased states are still poorly understood. The review further identifies the therapeutic potential of manipulating the interaction of NCS-1 with its many targets and suggests directions for future research on the role of NCS-1 in these disorders.

Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein

Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca2+-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca2+ with high affinity (Kd=1.46nM) and favourable Gibbs free energy (∆G=−12.4kcal/mol). The stoichiometry for Ca2+ bound to sbCRTAC2 at saturation indicated six Ca2+ ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca2+. Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell–cell and cell–matrix interactions.

Decreased hippocampal expression of calbindin D28K and cognitive impairment in MELAS

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited mitochondrial syndrome characterized by seizures, migrainous headaches, lactic acidosis, vomiting, and recurrent stroke-like episodes. Patients often suffer from cognitive dysfunction of unclear pathogenesis. In this study, we explored a possible link between cognitive dysfunction and hippocampal expression of calbindin D28KD (CB), a high affinity calcium-binding protein, in four MELAS patients, using post mortem hippocampal tissues. We found significantly reduced CB levels in all patients by immunohistochemistry, Western blot, and quantitative real-time PCR. Reduction in CB expression has been associated with aging and with neurodegenerative disorders, including Alzheimer's disease. We postulate that the reduced CB expression may play a role in the cognitive abnormalities associated with MELAS.

Two Frequenins in Drosophila: unveiling the evolutionary history of an unusual Neuronal Calcium Sensor (NCS) duplication

BackgroundDrosophila Frequenin (Frq), the homolog of the mammalian Neuronal Calcium Sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, Frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species.ResultsSurprisingly, we have found no amino acid replacements fixed across the twelve Drosophila species surveyed. In contrast, synonymous substitutions have been prevalent in the evolution of the coding region of frq1 and frq2, indicating the presence of strong functional constraints following gene duplication. Despite that, we have detected that significant evolutionary rate acceleration had occurred in Frq1 in early times from the duplication, in which positive selection (likely promoting functional diversification) had probably an important role. The analysis of sequence conservation and DNA topology at the non-coding regions of both genes has allowed the identification of DNA regions candidates to be cis-regulatory elements. The results reveal a possible mechanism of regulatory diversification between frq1 and frq2.ConclusionsThe presence of two Frequenins in Drosophila and the rapid accumulation of amino acid substitutions after gene duplication are very unusual features in the evolution of the Frq/NCS-1 subfamily. Here we show that the action of positive selection in concordance with some extent of regulatory diversification might explain these findings. Selected amino acid substitutions in Frq1 likely contributed to the functional divergence between the two duplicates, which, in turn, should have diverged in their regulation by Ecdysone-induced early genes.

The classical and a non-classical pathways associated with NF-κB are involved in estrogen-medicated regulation of Calbindin-D9k gene in rat pituitary cells

Calbindin-D9k (CaBP-9k) is a high affinity calcium binding protein that is highly expressed in the duodenum, kidney, uterus, and pituitary glands. Previous studies have shown that CaBP-9k expression is regulated by several steroid hormones, such as 1,25-dihydroxyvitamin D3, glucocorticoids, 17β-estradiol (E2), and progesterone (P4), in a tissue-specific manner. However, the promoter elements that mediate transcriptional regulation by these steroid hormones are not clearly understood, mainly due to the lack of an appropriate cell line expressing CaBP-9k. Recently it was shown that CaBP-9k was constitutively expressed in the rat pituitary gland, and is expressed in an E2-dependent manner in a pituitary gland tumor-derived cell line, GH3. In the current study, we examined the activity of the estrogen responsive element (ERE) in rat CaBP-9k gene in GH3 cells, using a luciferase gene reporter assay, electrophoretic mobility shift assay, and mutagenesis. A nuclear factor-κB (NF-κB) binding site in the CaBP-9k promoter region was identified (nucleotides −848 to −834 from the transcriptional start site), and we demonstrated that addition of an NF-κB blocker to GH3 cells reduced E2-induced CaBP-9k transcription. In the present study, we further showed a previously reported imperfect ERE (nucleotides +51 to +65) between exon I and intron A of CaBP-9k, indicating that the interaction of estrogen receptor (ER) α with this region is involved in the regulation of CaBP-9k promoter activity and its expression. Taken together, these results suggest that in GH3 cells, both the classical ERα-ERE pathway and a non-classical pathway involving NF-κB are involved in E2-mediatd regulation of CaBP-9k expression in the pituitary gland.

Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus

The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.

Calbindin-D28k and calcium sensing receptor cooperate in MCF-7 human breast cancer cells

The metastases of breast cancer cells to bone result in osteolysis and release of Ca2+. Ca2+ as a primary signal transducer can regulate the expression patterns of cell signaling systems. The extracellular calcium ion concentration sensing receptor CaR is a 123 kDa G-protein coupled membrane protein that resides within caveolin-rich regions as a dimer. CaR is involved in regulating several cellular processes such as proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a 28 kDa high affinity calcium-binding protein and it is involved in regulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to extracellular calcium ion concentration, [Ca2+]o, and neomycin sulfate, and spatial interactions of CaR with calbindin-D28k in MCF-7 human breast cancer cells were studied. Fura-2 loaded MCF-7 cells were exposed to increasing concentrations of CaCl2 or neomycin sulfate and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 or neomycin sulfate caused an increase in [Ca2+]i. The normalized dose response curves fitting yielded Hill co-efficient values of 4.32+/-0.63 and 1.49+/-0.14 for Ca2+ and neomycin sulfate respectively, thus indicating highly co-operative, 4-5 binding sites for Ca2+ and 1-2 binding site(s) for neomycin sulfate on CaR. The EC50 values were 21+/-1.6 mM and 43+/-3.5 micro M for CaCl2 and neomycin sulfate respectively. The confocal microscopy data, obtained by using a highly sensitive tyramide signal amplification technology for immunofluorescence detection, showed CaR and calbindin-D28k were co-localized when cells were exposed to 200 micro M neomycin sulfate, whereas in control cells there was no co-localization of these two proteins. We hypothesize that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.

Ca2+ sensing receptor activation by CaCl2 increases [Ca2+]i resulting in enhanced spatial interactions with calbindin-D28k protein

The extracellular calcium ion concentration, [Ca2+]o, sensing receptor CaR is a G-protein coupled membrane receptor and it is involved in regulating cell proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a high affinity calcium-binding protein that plays important roles in modulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to [Ca2+]o and spatial interactions of CaR with calbindin-D28k in a distal tubule-like renal cell line are described. Fura-2 loaded Madin Darby bovine kidney (MDBK) cells were exposed to increasing concentrations of CaCl2 and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 caused continual increase in [Ca2+]i. The thapsigargin induced increase in [Ca2+]i observed in basal medium was eliminated by pretreatment of MDBK cells with 10 mM CaCl2 thereby suggesting the involvement of endoplasmic reticulum Ca2+ stores in increasing the [Ca2+]i. CaR was localized in the plasma membrane by using confocal microscopy. The confocal microscopy data also showed CaR and calbindin-D28k were co-localized when cells were exposed to 40 mM CaCl2. We postulate that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.

Parvalbumin Expression Is Downregulated in Rat Fast-Twitch Skeletal Muscles during Aging

In this study, the protein expression profile of extensor digitorum longous (EDL) and Soleus (SOL) muscles, representing fast- and slow-twitch skeletal muscles, respectively, was established using high resolution two-dimensional electrophoresis (2-DE). One protein spot was found uniquely expressed in EDL muscle. N-terminal sequence analysis identified the protein as parvalbumin. Parvalbumin is a high affinity calcium binding protein that regulates muscle contraction and relaxation. Our experiments revealed that parvalbumin expression in EDL muscle was down-regulated during aging. In addition, high-intensity exercise could reverse this age-related change. Soleus muscles do not normally express parvalbumin, but high-intensity exercise could ectopically induce its expression in both young and old SOL muscles. We have also confirmed our 2-DE findings by immunohistochemistry on muscle sections. Our results suggest that high-intensity training could be used to improve muscle functions during aging because parvalbumin play an important role in regulating skeletal muscle contraction and relaxation.

Calreticulin and Calsequestrin are Differentially Distributed in Canine Heart

Cardiac sarcoplasmic reticulum (SR) consists of three continuous yet distinct regions: longitudinal, corbular, and junctional. Ca2+uptake, catalyzed by the Ca2+-dependent ATPase, is thought to occur throughout the SR whereas Ca2+release occurs in the region of the junctional SR. In the SR, Ca2+is stored in a complex with Ca2+-binding proteins such as calsequestrin. In the present study, the distribution of the high-affinity calcium-binding protein, calreticulin, in canine cardiac SR was determined and compared with the distribution of other SR proteins. Three types of distribution were observed. Many proteins, including the Ca2+-ATPase and two mannose-containing glycoproteins (52 and 131 kDa), were present in all subfractions. These proteins were absent or depleted from the sarcolemma-enriched fraction. The ryanodine receptor/Ca2+release channel and calsequestrin were localized to the junctional SR. Calreticulin immunoreactivity was detected in fractions containing longitudinal SR but not in fractions enriched in sarcolemma or junctional SR. Hence calreticulin is present in a unique vesicular fraction and the Ca2+-binding proteins calreticulin and calsequestrin display different patterns of distribution in canine heart.

Modelling oscillations of calcium and endoplasmic reticulum transmembrane potential: Role of the signalling and buffering proteins and of the size of the Ca 2+ sequestering ER subcompartments

Intracellular calcium oscillations provide a natural clock that may be of crucial importance for the timing of many cellular processes. Elucidating of the mechanisms underlying these oscillations is of particular interest. The theoretical description presented here extends existing models of calcium oscillations by allowing for two types of proteins differing in their calcium-binding properties. This model reflects experimental findings by considering both a fast calcium-binding process to low-affinity protein binding sites such as found in the N-domains of calmodulin or troponin C and a class of high-affinity calcium binding proteins with slow binding kinetics (e.g., parvalbumin or the C-domains of calmodulin and troponin C). Furthermore, recalling that calcium is mainly stored in small subcompartments of the ER, it is argued that only a small fraction of its overall volume participates in the rapid release and uptake of calcium. The effect of the size of this fraction is studied. The hypothesis saying that any electric potential difference across the ER membrane would be dissipated by the highly permeant ions is critically examined by an analytical estimation based on the electroneutrality condition and by numerical integration of the complete model equations. It is predicted theoretically that the transmembrane potential of the ER calcium stores, which is up to now virtually impossible to determine in experiment, builds up in the millivolt range at physiological concentrations of monovalent ions. The phenomenology of oscillations is studied by numerical integration. The model reproduces experimentally observed values of frequency and amplitude as well as the typical spike-like shape of oscillations. The model reveals also the time course of a shift of the bound Ca 2+ population from the low-affinity binding sites to the high-affinity binding sites.

Increased calreticulin stability in differentiated NG-108-15 cells correlates with resistance to apoptosis induced by antisense treatment

Since its first identification as a high-affinity calcium-binding protein over two decades ago [T.J. Ostwald and D.H. MacLennan, Isolation of a high-affinity calcium-binding protein from sarcoplasmic reticulum, J. Biol. Chem., 249 (1974) 974–979], calreticulin has become recognized as a multifunctional protein involved in a wide variety of cellular processes. We have previously shown that it has a protective function in Ca 2+-mediated cell death [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca 2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635–28639]. We report here that in NG-108-15 neuroblastoma×glioma hybrid cells, calreticulin protein levels increase markedly when these cells are induced to differentiate by treating them with N, N-dibutyryl cAMP (db-cAMP). We demonstrate that the reason for this increase is mostly due to a large increase in the turnover time of calreticulin in differentiated cells. We also show that a calreticulin antisense oligonucleotide, CrtAS1, previously described by Liu and co-workers [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca 2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635–28639] causes cell death in undifferentiated NG-108-15 cells when antisense treatment is extended for more than 24 h. This effect is not seen in NG-108-15 cells that have been induced to differentiate with db-cAMP until the cells have been treated with antisense for more than 4 days, due to the increased stability of Crt in these cells. Our results indicate that the mechanism by which these cells die is likely to be apoptosis.

A subpopulation of WIL-2 cell calreticulin molecules is associated with RO/SS-A ribonucleoprotein particles.

A subpopulation of human calreticulin (CR) molecules that is reactive with human Ro/SS-A autoimmune sera was identified in a nucleic acid- enriched Wil-2 cell fraction derived by anion exchange column chromatography. Further resolution of this fraction by gel filtration size separation demonstrated that the appearance of CR (true mol. weight 46 kD) coincided with the emergence of Ro/SS-A ribonucleoprotein (mol. weight > 250 kD) antigenic activity and increasing 260 nm ultraviolet absorbance. This high nucleic acid fraction could be further partitioned into four small RNA-containing Ro/SS-A antigenic subfractions by a second passage over the anion exchange column. CR was enriched in one subfraction and present in the other three subfractions as well. No CR was found in the RNA-free fraction of the repartition eluate. These results represent the first direct demonstration that CR, a high-affinity calcium binding protein, exists in a form that is directly associated with all four varieties of native, human Ro/SS-A ribonucleoprotein particles (hY1-5).

Abundant calcium homeostasis machinery in rat dental enamel cells. Up-regulation of calcium store proteins during enamel mineralization implicates the endoplasmic reticulum in calcium transcytosis.

Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium-binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium-binding proteins in enamel cells exceeded that in brain and other established calcium-oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high-affinity calcium-binding protein (12 kDa, distinguished from alpha-parvalbumin) that was up-regulated during maturation, but its low abundance (0.02% of soluble protein) precluded a major calcium transport or cytoprotective role. Two low-affinity calcium-binding proteins up-regulated during maturation (by 1.8-fold and 2.1-fold respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67-fold higher than that of the principal cytosolic calcium-binding protein. 28-kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was highly expressed during maturation, supported the novel hypothesis that non-mitochondrial calcium stores play a major role in transcellular calcium transport. (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium-binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non-mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium-transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium-oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.

Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa.

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.

Calbindin28kDa and Calmodulin are Hyperabundant in Rat Dental Enamel Cells. Identification of the Protein Phosphatase Calcineurin as a Principal Calmodulin Target and of a Secretion-Related Role for Calbindin28kDa

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin2gkDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33 % of total (SDS-soluble) calbindin2SkDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskele-tal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDil and calcineurin demonstrated here provides the first molecular evidence that enamel cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.

Calmodulin regulates fast axonal transport of squid axoplasm organelles

The role of calmodulin (CaM) in organelle motility (fast axonal transport) in the axoplasm of the squid giant axon was evaluated directly using video-enhanced microscopy. Addition of 6 μM CaM to extruded squid axoplasm produced a 2.6-fold increase in the number of organelles moving per minute per unit area of axoplasm. When lower concentrations of CaM, including physiological concentration (2 μg/ml), were added to extruded axoplasm, the number of organelles moving was equally increased. CaM had no significant effect on the mean velocity of organelle translocations. The stimulatory effect of CaM was reduced significantly by the CaM inhibitors melittin (36 μM) and trifluoperazine (50 μM). Parvalbumin, a high-affinity calcium binding protein, did not stimulate motile activity. These results suggest that CaM is a positive regulator of fast axonal transport. At the molecular level, this regulation may involve microtubule-and/or actin-based motor proteins. Several possible molecular mechanisms are proposed.

Calcium-binding properties of SSP-5, the Streptococcus gordonii M5 receptor for salivary agglutinin

Streptococcus gordonii M5 expresses a lectin on its surface (SSP-5) which binds to human salivary agglutinin (SAG). This interaction requires sialic acid residues of SAG and divalent cations and may mediate the colonization of oral tissues by this organism. In this report, we show that the binding of SAG to SSP-5 requires calcium and that SSP-5 is a high-affinity calcium-binding protein. SAG-mediated aggregation of S. gordonii M5 was inhibited by 1 mM EDTA, and the restoration of aggregation occurred only upon the readdition of calcium. To ascertain the level at which calcium exerts its effects, the calcium-binding properties of SSP-5 were evaluated by using a 45Ca binding assay. In addition, a kinetic analysis of calcium binding was carried out by using fura2, a fluorescent calcium-binding dye. These analyses showed that SSP-5 is a high-affinity calcium-binding protein that binds 1 mol of calcium per mol of protein and has a dissociation constant of 0.45 +/- 0.2 microM. The calcium-binding capacity of SSP-5 was also calculated independently to be 1.0 +/- 0.2 mol of Ca per mol of SSP-5 by column chromatography on Sephadex G-25 equilibrated with 10 microM 45Ca. To localize the calcium binding site of SSP-5, a series of C-terminal deletion mutants were expressed in Escherichia coli and evaluated for calcium-binding activity. Deletion of the 250 C-terminal residues of SSP-5 had little effect on calcium binding. However, deletion of residues 1168 to 1250 resulted in the loss of calcium-binding activity, suggesting that this region is important for calcium binding by SSP-5.