Isolation of the calcium-binding domain of carp parvalbumin

Carp parvalbumin has two calcium-binding domains with a similar three-dimensional structure. Using the tryptic hydrolysis at the arginine residue in position 75, it was possible to split off one calcium-binding domain. All lysine residues were protected by maleic groups which were removed at the final stage. The domain (with a peptide thirty-three residues) isolated by ion-exchange chromatography and gel filtration does not have a secondary structure in a solution and is unable to bind calcium.     

Tetrahymena calcium-binding protein is indeed a calmodulin

We previously isolated a Ca2+-binding protein from a ciliate, Tetrahymena, and designated it as TCBP (Tetrahymena Ca2+-binding protein). The present paper reports that TCBP, which has two high affinity Ca2+-binding sites (Kd=4.6 X 10(-6) M), could activate porcine brain cyclic nucleotide phosphodiesterase at a concentration of over 10(-6) M free Ca2+, with the same mode of activation as that of authentic (porcine brain) calmodulin. In addition, the amino acid composition of TCBP was essentially the same as that of brain calmodulin. Therefore, we conclude that TCBP as an activator of Tetrahymena guanylate cyclase is indeed a calmodulin.     

Microtubule-associated protein, MAP2, is a calcium-binding protein

Calcium has been suggested to be an important element in the regulation of microtubule dynamics 'in vivo'. In this report we have analyzed the possibility that microtubule-associated protein 2 (MAP2) binds calcium. MAP2 was blue-stained with the cationic carbocyanine dye 'stains-all' in a similar way to that of calcium-binding proteins and bound 45Ca as estimated from dot-blotting experiments. The calcium-binding characteristics of MAP2, determined by equilibrium dialysis, indicated that MAP2 bound about 3 mol (n = 2.9 +/- 0.4) of calcium per mol of protein (Kd = (0.9 +/- 0.2).10(-5) M). Analysis of the Scatchard plots from equilibrium dialysis and dot-blot assays indicated that MAP2 also presented low-affinity calcium-binding sites (Kd = (0.3 +/- 0.2).10(-4) M). Incubation of nitrocellulose blots of proteolytically digested MAP2 with 45Ca indicated that the calcium-binding sites were located in the region that is not involved in the interaction with tubulin (projection region).     

Terbium replacement of calcium in carp muscle calcium-binding parvalbumin: an x-ray crystallographic study.

By use of an ethanol/phenol mixture for stopping the pulse, joining of the labelled P2 short DNA chains during the subsequent operation is abolished more completely than with the ice and KCN mixture used previously (Kainuma-Kuroda &; Okazaki, 1975). After stopping a brief [³H]thymidine pulse with this mixture, 60 to 65% of the radioactivity incorporated is recovered in the short chain fraction, while the rest is in DNA chains of one genome length or longer. Hybridization with the complementary phage DNA strands clearly indicates the presence of a small amount of nascent short chains of the L-strand. However, even after a very brief pulse, two-thirds of the pulse label incorporated into the L-strand is in DNA chains of one genome length or longer. If P2-infected cells of a polA^ts strain are pulse-labelled after transfer to a restrictive temperature, virtually all the label incorporated is found in short DNA chains. These short DNA chains, accumulated during inhibition of host DNA polymerase I, contain equal amounts of H and L-strand components. From these findings it is concluded that both strands of P2 DNA are replicated by the discontinuous mechanism but that the rate of joining of the short chains is very much faster in the L-strand than in the H-strand.     

Intestinal calcium-binding protein

Summary The vitamin D-dependent calcium-binding protein (CaBP) was studied in relation to the age of the cell, in isolated epithelial cell populations removed from rat duodenum. Alkaline phosphatase and thymidine kinase activities were used as markers to characterize differentiated villus cells and undifferentiated (mitotically active) crypt cells, respectively. CaBP distribution along the length of the villus, as established by radioimmunoassay, appears as a gradient increasing from the crypt to the tip of the villus. CaBP concentration in cells is shown to be (i) negatively correlated with the thymidine kinase activity of cells, and (ii) positively correlated with the alkaline phosphatase activity of cells. This indicates that CaBP is absent in crypt cells and appears in differentiated cells with the development of the brush border. Thus CaBP, like alkaline phosphatase, can be considered as an indicator of enterocyte maturation. These data were also confirmed by studying the cellular localization of the protein. In addition both indirect immunofluorescence and immunoperoxidase staining methods reveal that antibody against CaBP decorates the terminal web, but not the microvilli of the brush border of mature absorptive cells. The results suggest that CaBP may act as a modulator of some Ca²⁺-mediated biochemical processes at the level of the enterocyte brush border.Portions of this work were presented at the Fourth International Workshop on Calcified Tissues, Israel (March 1980)     

Distribution of calcium-binding proteins parvalbumin and calbindin in the pigeon telencephalic auditory center

Immunoreactivity for calcium-binding proteins parvalbumin (PV) and calbindin (CB) was studied in the pigeon (Columba livia) telencephalic auditory center. All its regions displayed overlapping distribution patterns of PV and CB immunoreactivity, although in the central (L2) vs. peripheral (L1, L3, CMM) layers they were dissimilar. L2 and the inner L1 sublayer (L1i) were distinguished by a higher immunoreactivity of neuropil for both proteins and the presence (in L2) of numerous small densely packed granular-type cells: heavily stained PV-ir and, as a rule, poorly stained CB-ir neurons. In Lli, the number of neurons and the density of neuropil immunoreactive to both proteins decreased. The outer L1 sublayer (L1e) as well as L3 and CMM were characterized by a generally lesser density and irregular distribution of immunoreactive neuropil and a heterogenous repertoire of PV-ir and CB-ir neurons referring to diverse morphological types, with an increased number of large multipolar cells. The differences in PV and CB immunoreactivity among different regions of the pigeon telencephalic auditory center revealed the similarity of the latter to the laminar auditory cortex in mammals.     

Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis.

The structure of carp muscle calcium-binding parvalbumin has been refined to an overall residual, ($\text{Σ¦F}{}_0\text{− F}{}_{\mathrm{c}}\text{¦}\text{Σ¦F}{}_0\text{¦}$), of 0.25 by a combination of model building and difference Fourier analyses. The atomic positions were allowed to vary up to 0.25 Å from their idealized positions; individual temperature factors ranged from 2 to 150; and 138 solvent peaks were included in this final structure factor calculation. The effects of varying these parameters were analyzed. The treatment of low-order reflections and the influence of solvent have been analyzed in terms of Babinet's principle. These procedures and results are applicable to other protein refinement problems. The errors in co-ordinates are estimated to range from 0.15 Å for the Ca²⁺ ions to 0.30 Å for internal side chain atoms.A van der Waals' radii study indicates that 42% of the crystal volume is occupied by solvent. There are 16 intermolecular contacts. Of the 108 main chain peptide hydrogen atoms, 15 are neither in contact with solvent nor involved in hydrogen bonds. These “lost” hydrogen bonds are considered to be important to the proposed model of function.     

A novel calcium-binding protein is associated with tau proteins in tauopathy

Tauopathies are a group of neurological disorders characterized by the presence of intraneuronal hyperphosphorylated and filamentous tau. Mutations in the tau gene have been found in kindred with tauopathy. The expression of the human tau mutant in transgenic mice induced neurodegeneration, indicating that tau plays a central pathological role. However, the molecular mechanism leading to tau-mediated neurodegeneration is poorly understood. To gain insights into the role that tau plays in neurodegeneration, human tau proteins were immunoprecipitated from brain lysates of the tauopathy mouse model JNPL3, which develops neurodegeneration in age-dependent manner. In the present work, a novel EF-hand domain-containing protein was found associated with tau proteins in brain lysate of 12-month-old JNPL3 mice. The association between tau proteins and the novel identified protein appears to be induced by the neurodegeneration process as these two proteins were not found associated in young JNPL3 mice. Consistently, the novel protein co-purified with the pathological sarkosyl insoluble tau in terminally ill JNPL3 mice. Calcium-binding assays demonstrated that this protein binds calcium effectively. Finally, the association between tau and the novel calcium-binding protein is conserved in human and enriched in Alzheimer's disease brain. Taken together, the identification of a novel calcium-binding protein associated with tau protein in terminally ill tauopathy mouse model and its confirmation in human brain lysate suggests that this association may play an important physiological and/or pathological role.     

Distribution of calcium-binding proteins parvalbumin and calbindin in the thalamic auditory center in pigeons

Distribution of calcium-binding proteins (CaBPr) parvalbumin (PV) and calbindin (CB) in the thalamic auditory center (nucleus ovoidalis, Ov) was studied in the pigeon (Columba livia). Two parts of Ov were distinguished on the basis of their cytoarchitectonics and distribution of PV and CB immunoreactivity. The central lemniscal region (core, nCe) contains both dense PV-ir neuropil and PV-ir neurons overlapped with scant CB-ir neuropil and weaker stained CB-ir neurons. The peripheral extralemniscal region (belt), consisting of peri/paraovoidal nuclei (Ovl, Ovm, SPO), contains only CB-ir neuropil and strongly stained CB-ir neurons morphologically differing from CB-ir neurons in the nCe. A comparative analysis of our data on the distribution of PV and CB immunoreactivity in the thalamic auditory relay center in pigeons and related literature data obtained on other avian, reptilian and mammalian species indicates high evolutionary conservatism of its extralemniscal region across all sauropside amniotеs and mammals in contrast to plasticity of its central lemniscal region due to adaptive, ecologically dependent transformations during the evolution.     

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.     

Calexcitin B is a new member of the sarcoplasmic calcium-binding protein family

Calexcitin (CE) is a calcium sensor protein that has been implicated in associative learning. The CE gene was previously cloned from the long-finned squid, Loligo pealei, and the gene product was shown to bind GTP and modulate K(+) channels and ryanodine receptors in a Ca(2+)-dependent manner. We cloned a new gene from L. pealei, which encodes a CE-like protein, here named calexcitin B (CE(B)). CE(B) has 95% amino acid identity to the original form. Our sequence analyses indicate that CEs are homologous to the sarcoplasmic calcium-binding protein subfamily of the EF-hand superfamily. Far and near UV circular dichroism and nuclear magnetic resonance studies demonstrate that CE(B) binds Ca(2+) and undergoes a conformational change. CE(B) is phosphorylated by protein kinase C, but not by casein kinase II. CE(B) does not bind GTP. Western blot experiments using polyclonal antibodies generated against CE(B) showed that CE(B) is expressed in the L. pealei optic lobe. Taken together, the neuronal protein CE represents the first example of a Ca(2+) sensor in the sarcoplasmic calcium-binding protein family.     

The calcium-binding activity of a vacuole-associated,dehydrin-like protein is regulated by phosphorylation

A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 +/- 0.142 mM and 0.64 +/- 0.08 mM, and has an estimated 24.7 +/- 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.     

Observations on the binding of lanthanides and calcium to vitamin D-dependent chick intestinal calcium-binding protein. Implications regarding calcium-binding protein function

The binding of calcium and terbium to purified chick vitamin D-dependent intestinal calcium-binding protein was studied by terbium fluorescence, circular dichroism, and intrinsic protein fluorescence techniques. Calcium-binding protein bound, with high affinity, at least 3 mol of terbium/mol of protein; numerous low affinity terbium-binding sites were also noted. The three highest affinity sites were resolved into one very high affinity site (site A) and two other sites (sites B and C) with slightly lower affinity. Resonance energy transfer from tryptophan residues to terbium occurred only with site A. This site was filled before sites B and C. Competition experiments in which calcium was used to displace terbium bound to the protein showed that larger amounts of calcium were needed to displace terbium from site A than from sites B and C. Energy transfer from terbium to holmium indicated that the terbium-binding sites (B and C) were located close to each other (about 7-12 A) but were distant (greater than 12 A) from site A. The addition of EDTA to calcium-binding protein resulted in a 25% decrease in intrinsic protein fluorescence, suggesting a conformational change in the protein. The titration of EDTA-treated calcium-binding protein with calcium resulted in recovery of intrinsic protein fluorescence. A reversible calcium-dependent change in the ellipticity of calcium-binding protein in circular dichroism experiments was also seen. These observed properties suggest that vitamin D-dependent chick intestinal calcium-binding protein behaves in a manner similar to other well-known calcium-binding regulatory proteins.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.     

Recoverin, a novel calcium-binding protein from vertebrate photoreceptors.

Photoreceptor guanylyl cyclase activity is modulated by an endogenous calcium-binding protein called recoverin. A modified isolation procedure for recoverin using gel-filtration chromatography instead of a heat denaturation step is presented. The elution volume of recoverin corresponds to a monomer. Recoverin exhibits a calcium-dependent mobility shift in a native gel electrophoresis. Isoelectric focusing revealed a pI of 5.25. No subspecies of recoverin were detected.     

Neurocalcin: a novel calcium-binding protein from bovine brain.

A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain.