Soluble Calcium-binding Protein Research Articles

P-5 A soluble resistance related calcium binding protein (sorcin) up-regulates in the multi-drug resistant advanced gastric cancer patients

P-5 A soluble resistance related calcium binding protein (sorcin) up-regulates in the multi-drug resistant advanced gastric cancer patients

Label-free quantitative proteomic analysis of insect larval and metamorphic molts

BackgroundMolting is an essential biological process occurring characteristic times throughout the life cycle of holometabolous insects. However, it is not clear how insects determine the direction of molting to remain status quo or to initiate metamorphosis. To explore the functional factors that determine the direction of molts, liquid chromatography-mass spectrometry was used to identify the molecules involved in larval and metamorphic molting, and the differentially expressed proteins (DEPs) were compared in the two processes.ResultsThere were 321 and 1140 DEPs identified in larval and metamorphic molting process, respectively. Bioinformatics analyses show that the amino sugar pathway was up-regulated in both processes. The up-regulated protease contributed to the metamorphosis. In addition, several proteins with different expression patterns in larval-larval and larval-pupal transitions, including Endochitinase, GRIM-19 (Genes associated with retinoid-IFN-induced mortality-19), IDE (Insulin-degrading enzyme), Sorcin (Soluble resistance related calcium binding protein), OBP (Odorant-binding protein-2 precursor), TRAP1(Tumor necrosis factor receptor associated protein-1), etc., were further identified by parallel reaction monitoring, which may play diverse functions in larval-larval and larval-pupal transitions.ConclusionsThese results provide a proteomic insight into molecules involved in larval and metamorphic molts, and will likely improve the current understanding of determination of direction of molts.

Links of pathogenesis of pre- and perinatal damage of the children’s brain

A correlation analysis was made of the relationships between the structural changes in the brain and the content of idiotypic, anti-idiotypic antibodies to the soluble calcium-binding protein of the nervous tissue S100 in the serum of 318 children of the frst year of life with consequences of pre- and perinatal brain damage. We established the conjugation between the structural pathology of the brain, represented by cystic-atrophic changes, calcifcations, glia in the brain substance, periventricular leukomalacia, periventricular cysts, expansion of the ventricular system, congenital malformations of the brain, and the content of idiotypic, anti-idiotypic antibodies to the S100 protein in the serum blood of children 3, 6, 9, 12 months old. The concentration of idiotypic and anti-idiotypic antibodies to the protein of the nervous tissue S100 in the serum reflects the severity of destructive processes in the brain substance. Autoimmune processes are one of the mechanisms that lead to the structural damage to the brain in children with adverse pre- and perinatal factors.

Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency.

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 11 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

Age-dependent Expression of S100β in the Brain of Mice

S100beta is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100beta to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100beta in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT-PCR and Western blotting experiments show that the expression of S100beta gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100beta follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100beta is important for brain development and establishment of proper brain functions. Up-regulation of S100beta in old age may have some role in development of age-related pathological systems in the brain.

Variation of Sorcin's Expression in the Reversion of Multidrug Resistance of K562/A02 Leukemic Line with Tetrandrine.

Objective This paper aims to investigate the reversal multiple of daunomycin(DNR) with different concentration of tetrandrine(tet) at different time, to study the variation of Soluble resistance related calcium binding protein (sorcin)’s expression in the reversion of multidrug resistance of K562/A02 leukemic line with different concentration of tet at different time, and to provide new theoretic evidence for the clinical application of tet as resistence modifying agents. Method The reversal multiple of DNR with different concentration of tet at different time was assayed by MTT (concentration of tet is 0.5mg/l or1mg/l or 2mg/l, incubation time is 48h or 72h). The variation of the gene’s expression of sorcin with different concentration of tet at different time wasassayed by RT-PCR(grouping like MTT). Result MTT analysis demonstrated that the reversal concentration was 1.81(0.5mg/l,48h),3.62(1.0mg/l,48h),6.14(2.0mg/l,48h),2.8(0.5mg/l,72h),(1.0mg/l,48h),7.12(2.0mg/l,72h) respectively. RT-PCR analysis demonstrated that sorcin gene was lowly displayed in K562 cells and highly displayed in K562/A02 cells. The variation was first accentuation and then attenuation with the concentration accrescence of tet, this tendency had no obviously difference between 48h and 72h. Conclusion 1 The result of the experiment shows that tet may reverse multidrug resistance of K562/A02 cells by the down regulation of the expression of sorcin gene and proteinum; 2. It may have the concentration dependence and the time dependence in the reversion of multidrug resistance of K562/A02 cells with tet.

Temperature sensitivity of calcium binding for parvalbumins from Antarctic and temperate zone teleost fishes

Parvalbumin (PV) is a soluble calcium-binding protein that is especially abundant in fast-twitch muscles of fish and other lower vertebrates. Despite its prevalence in ectothermic taxa, few data address the effects of temperature on PV binding function. In this study, calcium dissociation constants ( K D) were measured as a function of temperature (0–25 °C) for PV from two Antarctic ( Gobionotothen gibberifrons and Chaenocephalus aceratus) and two temperate zone fish species ( Cyprinus carpio and Micropterus salmoides). Measurements by fluorometric competitive binding assay show that K D values for PVs from the Antarctic species were significantly higher at all assay temperatures and were less sensitive to temperature relative to carp and bass. However, estimates of K D are fundamentally similar for PVs from the Antarctic and temperate zone species when examined at their native physiological temperature. Variation in pH and ionic strength within a physiologically relevant range had only modest effects on K D. Thermodynamics of calcium binding to PV from G. gibberifrons and C. carpio was measured by isothermal microcalorimetry. When measured at 15 °C, the Gibbs free energy change (Δ G) was significantly greater for calcium binding to PV from G. gibberifrons than from carp (−43.4±1.5 kJ mol −1 and −46.6±3.0 kJ mol −1, respectively), and the relative contribution of entropy to Δ G for calcium binding to PV from the Antarctic species was about twice that of carp (Δ S=16.0±0.8 J °C −1 mol −1 for G. gibberifrons; Δ S=7.5±0.8 J °C −1 mol −1 for C. carpio).

Interaction of the proteasome S5a/Rpn10 multiubiquitin-binding protein and the 8 kDa calcium-binding protein of Schistosoma mansoni.

A distinct 8 kDa calcium-binding protein (CaBP) is preferentially expressed at the cercarial stage during the life-cycle of the schistosome. Available data indicate that this CaBP may be associated with tissue/organ remodelling (involving protein degradation and synthesis of new proteins) during transformation of the cercariae from free-living form in water to parasitic life in the vertebrate host. Many CaBP molecules (e.g. calmodulin) show Ca(++)-dependent interaction with target proteins and thus modulate their activity. Accordingly, the parasite 8 kDa CaBP was used as a probe to clone and identify putative target protein(s) directly by binding interaction. Screening of schistosome lambdagt11 expression library with radio-iodinated CaBP yielded several overlapping clones showing Ca(++)-dependent binding of the CaBP. Sequence analyses revealed that these clones encode the S5a/Rpn10 multiubiquitin-binding protein which is a component of the regulatory 19S subunit of the 26S proteasome. The schistosome molecule, designated SmS5a, is 420 amino acids long. The nearly full length molecule (Gln3-Ser420) as well as the amino terminal (N-S5a, Gln3-Gly200) and carboxyl-terminal (C-S5a, Asp225-Ser420) portions were synthesized in bacteria, purified, and antibodies to the parasite SmS5a were prepared. Interaction between SmS5a and the 8 kDa CaBP in a Ca(++)-dependent manner was found under various experimental conditions: CaBP-Sepharose bound soluble SmS5a, immobilized SmS5a bound soluble CaBP, and complex formation was found when both molecules were in solution. Furthermore, it was shown that the C-terminal portion of SmS5a, but not the N-terminal portion of the molecule, reacted with the CaBP. SmS5a synthesized in a cell-free system and Western blots revealed 2 species, conceivably corresponding to the naked molecule (approximately 50 kDa) and the molecule subjected to post-translational modification (approximately 70 kDa). The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

Titin–Actin Interaction in Mouse Myocardium: Passive Tension Modulation and Its Regulation by Calcium/S100A1

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK–actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK–actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK–actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin’s extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1–PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Prediction System for Dumbbell-Type Proteins: SOSUIdumbbell

A dumbbell-type (DT) protein comprises two globular domains and one helical segment connectingthese globular domains. DT protein plays an important role in the regulation of other proteins.Calmodulin (CaM), a typical DT protein, for example, is a soluble calcium-binding protein thatregulates several enzymes such as smooth muscle, myosin light-chain kinase and Ca

Quantification of the soluble calcium-binding protein (SCBP) in various muscle tissues of the terrestrial oligochaete Lumbricus terrestris L.

The soluble calcium-binding protein (SCBP) from the body wall muscle of Lumbricus terrestris can be assumed to be the counterpart of the high-affinity calcium-binding protein parvalbumin (PV) from vertebrate muscle, the physiological role of which remains to be established. It is proposed that these proteins could act as soluble relaxing factors, especially in fast contracting muscles (Gerday, 1988). One of the experimental findings supporting this idea is the correlation between the relaxation speed of a muscle and its content of PV. We have been prompted to perform a quantitative analysis on various types of muscle of the terrestrial oligochaete L. terrestris in order to verify the above mentioned correlation for the case of an invertebrate species. The results presented in this report indicate that the amount of SCBP within the body wall muscle is three orders of magnitude higher as compared to the gizzard muscle of L. terrestris.

A soluble calcium-binding protein (scbp) present in drosophila melanogaster and calliphora erythrocephala muscle cells

1. 1. Soluble calcium binding proteins (SCBP) were isolated from homogenates of whole flies, from the thorax and from muscles of Drosophila melanogaster and Calliphora erythrocephala. 2. 2. Crude preparations were obtained by extraction at low ionic strength, acid and heat treatment. The Drosophila protein was purified by gel filtration, hydrophobic interaction and ion exchange chromatography. In contrast to calmodulin the Drosophila SCBP did not bind to phenyl-Sepharose in a Ca 2+-dependent way. 3. 3. Both the Drosophila and Calliphora protein revealed identical properties. 4. 4. The apparent molecular mass of the SCBP is 24 kDa. Separation in urea-PAGE demonstrated the existence of two isoforms. 5. 5. The calcium-binding property was assured by a calcium dependent electrophoretic mobility shift and autoradiography of 45Ca 2+-incubated Western blots. 6. 6. The proteins are abundant in the thorax and were even detectable in crude extracts of various mascles (leg muscles and the extracoxal depressor). In contrast, in power muscles and in the thoracic ganglion the proteins could not be observed.

Measurement of facilitated calcium diffusion by a soluble calcium-binding protein

The flux of calcium through an aqueous compartment was determined in a flow-dialysis cell in which two dialysis membranes separated the middle aqueous compartment from two outer compartments. The contribution of convection to the total calcium flux was large but could be removed by addition of 1% agar. The flux of calcium through the gelled aqueous compartment agreed with theoretical expectations. The self-diffusion coefficient for calcium from these results was calculated to be 0.81·10 −5 cm 2·s −1. Carp parvalbumin significantly enhanced the calcium flux at 2.3·10 −6M free calcium. The calcium flux increased linearly with parvalbumin concentration. These observations are consistent with the hypothesis that the overall unidirectional calcium flux J is the sum of free calcium diffusion and protein-calcium diffusion: J = D[Ca] + D′[CaPr]. The value of D′, the self-diffusion coefficient for parvalbumin, was calculated from the flux data to be 13.7·10 −7 cm 2·s −1.

The soluble calcium-binding protein from muscle of the sandworm, Nereis virens.

Fast-acting muscle of the sandworm, Nereis virens, contains one soluble calcium-binding protein having a molecular weight close to 17 000 and occurring in the muscle at a concentration of approximately 0.1 mM. The protein binds two Ca2+ at equivalent sites with dissociation constant Kd = 6.4 X 10(-7) M. Its N-terminal amino acid is blocked by an N-acetyl group whereas glycine is the C-terminal residue. The comparison of the tryptic peptide map of this protein with those of the soluble calcium-binding protein from crayfish muscle, bovine brain calmodulin and rabbit skeletal muscle troponin C suggests that all of these proteins are homologous. Sandworm calcium-binding protein therefore belongs to the so-called cytosolic calcium EF-hand family. This protein is presumably the functional counterpart of vertebrate parvalbumin acting as soluble relaxing factor.

Intestinal membrane calcium-binding protein. Vitamin D-dependent membrane component of the intestinal calcium transport mechanism.

A particulate fraction of rat intestinal mucosal homogenates, termed the "calcium-binding complex," contains three vitamin D-dependent activities: calcium binding of high affinity, calcium-dependent adenosine triphosphatase, and p-nitrophenylphosphatase. These particulate activities vary concordantly with intestinal calcium transport, suggesting that they represent membrane components of the translocation mechanism. The particulate was solubilized with 1-butanol and the activities were resolved partially by gel filtration and by DEAE-cellulose and spheroidal hydroxyl-apatite column chromatography. The Ca-binding activity was separated from the enzymes and isolated as a protein of molecular weight approximately 200,000, as estimated by gel filtration in 0.1% Triton X-100. The membrane protein, named IMCal (intestinal membrane calcium-binding protein), was dissociated with sodium dodecyl sulfate to yield a monomer of molecular weight 20,500 which is clearly distinguishable from the soluble calcium-binding protein (molecular weight 11,500) of rat mucosa. The apparent dissociation constants of Ca2+ of IMCal and of the soluble calcium-binding protein were estimated as 0.37 microM and 2.25 microM, respectively. The vitamin D-dependent activities of the calcium-binding complex are present in isolated intestinal microvillus membranes and may mediate the translocation of calcium from the intestinal lumen to the cytosol.

Studies on the subcellular localization of the membrane-bound fraction of intestinal calcium-binding protein

Sorbitol density gradient centrifugation applied to intestinal mucosa homogenates resulted in a complete separation of soluble calcium-binding protein from the bound fraction of calcium-binding protein, providing further documentation of the bound pool of calcium-binding protein. The peak of the bound calcium-binding protein was not associated with the major peaks of any of the markers used, but was associated with minor peaks of alkaline phosphatase, RNA, and glucose-6-phosphatase. Lack of association of bound calcium-binding protein with (Na + + K +)-ATPase indicated that the bound calcium-binding protein is not on the basolateral membrane. Differential centrifugation fractionation indicated that the bound calcium-binding protein is not associated with nuclei or mitochondria. The bound calcium-binding protein also could not be detected in partially pyrified brush borders. Exclusion of the brush border and basolateral membranes as the location of the bound calcium-binding protein suggests an intracellular locate.

Intestinal Calcium-Binding Protein and Calcium Absorption in Cortisol-Treated Chicks: Effects of Vitamin D3and 1,25-Dihydroxyvitamin D3*

Vitamin D3 in rachitic chicks stimulates calcium absorption and induces the synthesis of two pools of intestinal calcium-binding protein (CaBP), one soluble and the other membrane bound. Cortisol acetate caused a decrease in calcium absorption which was accompanied by a decrease in soluble CaBP. Cortisol was similarly effective in 1,25-dihydroxyvitamin D3-dosed chicks, suggesting that the glucocorticoid effect was not entirely due to the defective synthesis of this metabolite. Ca absorption was directly correlated with soluble CaBP and alkaline phosphatase and inversely related to the ratio of bound to soluble CaBP. It was further observed that the slope of the Ca absorption vs. soluble CaBP regression line was greater in chicks given 1,25-dihyroxyvitamin D3 compared to those given vitamin D3, and this is interpreted to mean that another factor or condition, in addition to assayed concentrations of soluble CaBP, determines the degree of calcium absorption.

Evidence for a membrane-bound fraction of chick intestinal calcium-binding protein

After homogenization of intestinal mucosa from vitamin D-replete chicks and high speed centrifugation, the major proportion of the vitamin D-induced calcium-binding protein is present in the supernatant fraction. However, the centrifugate, after repeated washing, contains significant amounts of bound calcium-binding protein that can be solubilized by Triton X-100. The bound calcium-binding protein is identical to soluble calcium-binding protein by the criteria of immunological identity, electrophoretic mobility, and molecular size, as determined by gel filtration chromatography. The bound calcium-binding protein is only partially released by sonication, osmotic shock or by ribonuclease treatment Bound and soluble calcium-binding protein are not present in rachitic chick intestine. The addition of calcium-binding protein to rachitic mucosa prior to homogenization does not yield a Triton X-100 solubilizable form, indicating that bound calcium-binding protein in vitamin D-replete intestine is not due to adsorption of vesicular entrapmetn of soluble calcium-binding protein. The overall evidence suggests that part of the intestinal calcium-binding protein is membrane-bound.

Vitamin d-stimulated intestinal calcium absorption may not involve calcium-binding protein directly.

ONE of the earliest responses of chick intestine to cholecalciferol (vitamin D3) is the appearance of a soluble calcium-binding protein (CaBP) for which current evidence indicates a role in the intestinal absorption of calcium1,2. CaBP, which has never been detected in the intestine of cholecalciferol-deficient chickens, is synthesised de novo in response to cholecalciferol, apparently by the induction of the transcription of a specific mRNA for CaBP (ref. 3). In the chick CaBP biosynthesis is initiated after a lag of about 7 h after physiological doses of the vitamin. A correlation between the appearance of CaBP and the first detectable increase in calcium absorption in response to cholecalciferol was observed when CaBP was measured using immunological procedures4,5 but not using the less sensitive Chelex assay6. Consequently it has been generally accepted that CaBP plays a major role in chicks in cholecalciferol-stimulated calcium transport, but by a mechanism which has still to be described. These changes in intestinal CaBP levels and in calcium absorption are brought about by 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), the hormonal metabolite of D3. We report here the sequence and time scale of changes in calcium absorption and CaBP levels in the intestine in response to 1,25-(OH)2D3. By comparison with events observed after cholecalciferol administration, the changes in response to the hormone turn out to be much more rapid and to have different rates of decay, with calcium transport being stimulated before CaBP can be detected immunologically.

Calcium-binding proteins in electroplax and skeletal muscle comparison of the parvalbumin and phosphodiesterase activator protein of Electrophorus electricus

A soluble calcium-binding protein has been isolated from the red skeletal muscle of the electric eel ( Electrophorus electricus). The purification procedure involved ammonium sulfate precipitation, gel filtration on Sephadex G-75 in the presence of 45Ca 2+, and chromatography on QAE-Sephadex. This procedure resulted in the isolation of a protein which was homogeneous upon polyacrylamide gel electrophoresis. The calcium-binding protein was found to be a typical parvalbumin by the following criteria: (1) molecular weight of 11 000; (2) pl of 4.7; (3) 1.9 mol Ca 2+ bound per mol protein; K d of approx. 10 −7 M; (4) no detectable phosphorus; (5) amino acid composition included nine residues of phenylalanine, single arginine, and no tyrosine or tryptophan; (6) ε max at 259 nm; (7) 260 nm:280 nm absorbance ratio of 4.78. Only one parvalbumin could be detected in muscle. Immunoprecipitation assay revealed that the parvalbumin was a major soluble component of skeletal muscle (0.10 mg/mg soluble protein), but could not be detected in liver, kidney, brain, spleen, heart or electroplax. Comparison of the parvalbumin with a calcium-binding protein previously isolated from electroplax revealed that the two proteins were different as judged by a variety of chemical criteria. These results suggest that during embryological development of electroplax the parvalbumin is lost and that it is not required for the function of electric tissue.