Functional analysis of a subset of canine olfactory receptor genes

In this paper, we explored the level complexity of the combinatorial olfactory code that allows mammals with a repertoire of about thousand putatively active olfactory receptors encoded in their genomes to recognize and identify a much larger repertoire of odorant molecules. To that end, we cloned 38 canine OR genes belonging to the same OR gene family and transiently expressed them in a subclone of embryonic human kidney cells (HEK293) permanently expressing the G(olf) subunit. Using a Ca(2+) imaging approach, we established for example that as many as 26 out of the 38 cloned OR elicited a Ca(2+) response when exposed to octanal, whereas 10 responded to nonanal, other aldehydes providing intermediate responses. Altogether, these results demonstrated that the combinatorial code is quite complex in support to the highly developed sense of olfaction demonstrated by dogs.     

No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids

The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.     

Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges

Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.     

Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells)

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.     

The solution structure of a plant calmodulin and the CaM-binding domain of the vacuolar calcium-ATPase BCA1 reveals a new binding and activation mechanism

The type IIb class of plant Ca(2+)-ATPases contains a unique N-terminal extension that encompasses a calmodulin (CaM) binding domain and an auto-inhibitory domain. Binding of Ca(2+)-CaM to this region can release auto-inhibition and activates the calcium pump. Using multidimensional NMR spectroscopy, we have determined the solution structure of the complex of a plant CaM isoform with the CaM-binding domain of the well characterized Ca(2+)-ATPase BCA1 from cauliflower. The complex has a rather elongated structure in which the two lobes of CaM do not contact each other. The anchor residues Trp-23 and Ile-40 form a 1-8-18 interaction motif. Binding of Ca(2+)-CaM gives rise to the induction of two helical parts in this unique target peptide. The two helical portions are connected by a highly positively charged bend region, which represents a relatively fixed angle and positions the two lobes of CaM in an orientation that has not been seen before in any complex structure of calmodulin. The behavior of the complex was further characterized by heteronuclear NMR dynamics measurements of the isotope-labeled protein and peptide. These data suggest a unique calcium-driven activation mechanism for BCA1 and other plant Ca(2+)-ATPases that may also explain the action of calcium-CaM on some other target enzymes. Moreover, CaM activation of plant Ca(2+)-ATPases seems to occur in an organelle-specific manner.     

The role of the GX9GX3G motif in the gating of high voltage-activated Ca2+ channels

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A > G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.     

A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium

This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.     

Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction

Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin-binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.     

Tuning of a neuronal calcium sensor

Recoverin is a Ca(2+)-regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca(2+) sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca(2+) sensitivity.     

Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi

Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi. Evidences have shown that Ca(2+) may play an important regulatory role in trypanosomatid flagellar mobility. In these parasites, the response of the cell to variations of Ca(2+) levels is determined by a variety of calcium-modulated proteins. Starting from T. cruzi cDNA lambdagt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected. rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment. In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography. Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca(2+) binding. The Ca(2+) binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca(2+)-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K(a) of 3.3+/-0.5 (10(6)) and 1.9+/-0.2 (10(4)) M(-1). Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca(2+)-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor.     

N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.     

The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.     

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.     

刀鲚嗅觉受体基因MOR-51I2克隆、序列分析及组织表达

为研究刀鲚的嗅觉受体（Olfactory receptor,OR）是否参与其生殖洄游过程,利用基因组步移技术,从洄游型刀鲚（Coilia nasus）中克隆出嗅觉受体基因MOR-51I2的基因序列全长。该基因为单外显子结构,编码区长为999 bp。在3'UTR区域具有一段微卫星序列,以（AC）n为重复单位,并夹有若干T或G碱基,且在不同生态型间具有明显长度差异。MOR-51I2基因所编码的蛋白具有7次疏水性α-螺旋的跨膜结构,为G-蛋白偶联受体。MOR-51I2基因与已报道的其他鱼类的OR基因所编码的氨基酸序列的同源性在51%以上,其中与大西洋鲱（Clupea harengus）的OR51I2-like基因的同源性高达83%。经qRT-PCR分析显示,在定居型刀鲚中,MOR-51I2基因主要在嗅囊和性腺中表达,在肝脏、鳃、肌肉微弱表达,在心脏和眼睛中几乎不表达。其中,雌性嗅囊中的表达量约是雄性嗅囊中的2倍,是精巢和卵巢中的80-100倍。在洄游型刀鲚中,该基因在雄性嗅囊的表达量约是雌性嗅囊中的6倍。MOR-51I2基因在洄游型刀鲚的雌性嗅囊中的表达量约是定居型刀鲚的雌性嗅囊中的1/5,而在洄游型刀鲚的雄性嗅囊中的表达量却是定居型刀鲚的雄性嗅囊中的3倍。这些结果表明,MOR-51I2基因不但参与刀鲚的嗅觉功能,而且可能参与了刀鲚的性腺发育及生殖洄游过程,同时也可能与其生态型的分化相关。     

Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins

Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

The CorA Mg(2+) transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the second membrane domain

Salmonella typhimurium CorA is the archetypal member of the largest family of Mg(2+) transporters of the Bacteria and Archaea. It contains three transmembrane segments. There are no conserved charged residues within these segments indicating electrostatic interactions are not used in Mg(2+) transport through CorA. Previous mutagenesis studies of CorA revealed a single face of the third transmembrane segment that is important for Mg(2+) transport. In this study, we mutated hydroxyl-bearing and other conserved residues in the second transmembrane segment to identify residues involved in transport. Residues Ser(260), Thr(270), and Ser(274) appear to be important for transport and are oriented such that they would also line a face of an alpha-helix. In addition, the sequence (276)YGMNF(280), found in virtually all CorA homologues, is critical for CorA function because even conservative mutations are not tolerated at these residues. Finally, mutations of residues in the second transmembrane segment, unlike those in the third transmembrane segment, revealed cooperative behavior for the influx of Mg(2+). We conclude that the second transmembrane segment forms a major part of the Mg(2+) pore with the third transmembrane segment of CorA.     

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

Cloning and characterization of Xenopus dicalcin, a novel S100-like calcium-binding protein in Xenopus eggs.

To contribute to the study of the calcium-signaling mechanism of egg, we cloned and characterized a 26 kDa Ca(2+)-binding protein from Xenopus laevis eggs, a homologue of Rana catesbeiana dicalcin (renamed from p26olf) that was isolated from the olfactory epithelium. The primary structure of Xenopus dicalcin shows approximately 61% identity to that of Rana dicalcin and consists of two S100-like regions aligned in tandem, as seen in Rana dicalcin. Genomic Southern blot analysis indicated that Xenopus dicalcin is a unique orthologue of Rana dicalcin. Northern blot analysis showed that Xenopus dicalcin mRNA is expressed in Xenopus eggs and also in other tissues. These results indicated that Xenopus dicalcin is a novel S100-like Ca(2+)-binding protein in Xenopus eggs.