Calcium Binding Capacity Research Articles

Development and in vitro evaluation of doxorubicin and celecoxib co-loaded bone targeted nanoparticles

Bone metastasis is one of the major problems for the treatment of cancer. Doxorubicin (DOX) is a potential drug for the treatment of bone metastasis with a limited usage due to systemic side effects. Also most of the many studies showed that celecoxib (CXB), a non-steroidal anti-inflammatory drug, have synergistic activity with DOX against cancer but usage of this combination is limited due to cytotoxicity of these drugs. In this study, it is aimed to reduce systemic side effects of these two synergic molecules (DOX and CXB) and increase efficacy of treatment. For this purpose, DOX and CXB co-loaded alendronate coated PLGA nanoparticles were prepared, optimized and fully characterized. Then, efficacy of nanoparticles was investigated by cytotoxicity, the calcium binding capacity and hydroxyapatite affinity tests. Results showed that fixed dose combination has been successfully generated and encapsulation ratio of DOX and CXB controlled by experimental design. Additionally cytotoxicity and targeting efficiency of the formulation has been clearly demonstrated. In conclusion, this study suggests that DOX and CXB co-loaded PLGA nanoparticles could be a promising approach for the treatment of bone metastases.

In search of the best xenogeneic material for a paediatric conduit: an experimental study.

The development of calcification-resistant bioprosthetic materials is a very important challenge for paediatric surgery. The subcutaneous implantation in rats is the well-known first-stage model for this kind of research. Using this model, we aimed to compare calcification of the porcine aortic wall and bovine pericardium and jugular vein wall cross-linked with glutaraldehyde (GA) and ethylene glycol diglycidyl ether (DE). We also determined the efficacy of DE-preserved tissue modification with 2-(2-carboxyethylamino)ethylidene-1,1-bisphosphonic acid (CEABA). Three groups of each biomaterial were evaluated: GA-treated, DE-treated and DE + CEABA-treated. The microstructure of non-implanted biomaterials was assessed by light microscopy after Picro Mallory staining; the phosphorus content of the DE and DE + CEABA samples was assessed by atomic emission spectrometry. Samples were implanted subcutaneously into young rats for 10 and 60 days. The explant end-point included quantitative calcification assessment by atomic absorption spectrophotometry and light microscopy examination after von Kossa staining. All GA-treated biomaterials had a high calcium-binding capacity (>100 μg/mg dry tissue). DE preservation decreased the vein wall and pericardium calcium content by 4- and 40-fold, respectively, but was ineffective for the aortic wall. The calculated CEABA content was almost equal in the vein wall and pericardium (17.7 and 18.5 μM/g) and slightly less in the aortic wall (15 μM/g) (P = 0.011). CEABA effectively reduced mineralization in the DE aortic wall and DE pericardium to 10.1 (7.8-21.1) and 0.95 (0.57-1.38) μg/mg but had no effect in the DE vein wall. Mineralization in the GA- and DE-treated aortic and vein walls was predominantly associated with elastin. CEABA modification decreased elastin calcification but did not block it completely. Each xenogeneic material requires individual anticalcification strategy. DE + CEABA pretreatment demonstrates a high mineralization-blocking efficacy for the bovine pericardium and should be employed to further develop the paediatric pericardial conduit. Aortic wall calcification cannot be blocked completely using this strategy.

Synthesis and retarder mechanism study of a novel amphoteric composite high temperature-resistant retarder for oil well cement.

An amphoteric composite polymer (hereinafter referred to as PAADM) as a high temperature-resistant cement retarder was prepared by in situ intercalated polymerization method with 2-crylamido-2-methylpropanesulfonic acid (AMPS), acrylic acid (AA) and two diallyl dimethyl ammonium chloride (DMDAAC) as monomers, and modified montmorillonite as an active polymerization filler. The synthetic composite polymer was characterized by Fourier transform infrared spectroscopy (FT-IR), H nuclear magnetic resonance (H-NMR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). The results of the aforementioned characterization showed that the synthesized copolymer (PAADM) has an intercalation/exfoliation composite structure and excellent thermal stability. Performance evaluation evidenced that the cement slurry containing PAADM has good retarding property in the range of 120–200 °C, and demonstrated the rapid development of compressive strength under high temperature and low temperature conditions, this property could guarantee that the retarder PAADM could be applied to the construction of deep wells and long interval wells. Moreover, the retarding mechanism of PAADM was studied through calcium binding capacity, adsorption amount, zeta potential, XRD and SEM analysis, and it was found that “adsorption deposition” and “calcium complexation” should be responsible for this retarder delaying the hydration process of cement grains.

Preparation, characterization and calcium release evaluation in vitro of casein phosphopeptides-soluble dietary fibers copolymers as calcium delivery system

Delivery systems based on food-grade biopolymers are of particular interest for food applications. Here, we introduce a new calcium delivery system with a high calcium-binding capacity and controlled calcium release. The system integrates casein phosphopeptides (CPP) with soluble dietary fibers (SDF). The physicochemical properties, calcium-binding capacities, controlled calcium release properties and formation mechanisms of the prepared copolymers with different mass ratios of CPP/SDF after thermal treatment were studied. The results showed that CPP-SDF copolymers significantly improved the calcium binding capacity (P < .05) from 11.63% to 46.8% and increased the calcium phosphate deposition time. The SDF in copolymers protected CPP from gastric enzyme hydrolysis so that most of the calcium was released under intestinal conditions in a controlled manner. Structural characterizations demonstrated different copolymer conformational states, and more calcium-binding sites existed within the CPP-SDF copolymers. The results show that copolymers based on food-sourced polymers are potential candidates for oral calcium delivery.

A novel calcium-binding peptide from Antarctic krill protein hydrolysates and identification of binding sites of calcium-peptide complex

Trypsin was used for preparing peptides with high calcium-binding capacity from Antarctic krill. Hydroxyapatite chromatography (HAC), size-exclusion chromatography (SEC), and reversed phase high performance liquid chromatography (RP-HPLC) were used to capture and purify calcium-binding peptides. The peptide sequence was determined to be VLGYIQIR (N- to C-terminal, MW = 960.58 Da), using LTQ Orbitrap XL. According to the results of FTIR and mass spectrometry, chelating site of calcium ions may possibly involve the carbonal or amino groups of Gln, Ile and Arg residues. Molecular dynamic simulation showed the conformation of peptide was markedly varied, and the distance between calcium ion and Gln and Ile residues was changing all the time. However, the distance between calcium ion and carboxyl oxygen of arginine residues was not changed significantly from 2 ns to 100 ns. Identified peptide can be used as a novel calcium supplement.

Interaction mode of calcium-binding peptides and Caco-2 cell membrane

In our previous studies, soluble soybean protein hydrolysate (SPH)–calcium complexes were shown to promote the calcium uptake of Caco-2 cells. However, the calcium transport mode involved remains unknown. In this article, several experiments were carried out via cytological analysis to investigate the calcium transport mode of peptides with low calcium binding capacities (F1), peptides with high calcium-binding capacities (F2), and their separate calcium complexes (F1–Ca and F2–Ca) when interacting with cell membranes. The interaction between one of them and a cell membrane is the first step in intracellular transport, as indicated by fluorescence blue shift experiments and acrylamide quenching experiments. The results of zeta potential experiments showed that only the “charge neutralization” phenomenon occurs when the F1 peptide or F1–Ca complex interacts with cell membranes and thus cannot be transported into cells. On the contrary, in an F2 at high concentrations or F2–Ca complex, a “charge recovery” phenomenon occurs apart from “charge neutralization” and can thus be transported into cells through endocytosis.

Phosphorylation Modulates Ameloblastin Self-assembly and Ca 2+ Binding.

Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

Abstract 5866: t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites

Abstract t-Darpp, one of two protein isoforms encoded by the PPP1R1B gene, is overexpressed in gastric, esophageal, colon, prostate, and breast cancers; and confers resistance to trastuzumab in Her2+ breast cancer cells. To gain insight into the trastuzumab-resistance mechanism of t-Darpp, we studied its structure, oligomerization status, metal-binding properties, and sites of phosphorylation. Circular dichroism spectroscopy analysis showed that recombinant t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil character at 25°C. Upon mild heat treatment (50°C for five min) the secondary structure does not appreciably change. Metal ion-binding analyses through inductively coupled plasma-mass spectrometry and graphite furnace-atomic absorption indicated that t-Darpp co-purifies with calcium, but not other metals commonly found in biological systems. The T75 site, critical for t-Darpp activation of the Akt signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, native blue gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments showed that the majority of t-Darpp (calculated mass = 19 kDa) exists as a monomer, but forms low levels (&amp;lt; 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a relatively large Stokes radius of 4.4 nm, suggesting an elongated structure. In summary, this study indicates that t-Darpp is an elongated, mild-heat-stable, and monomeric calcium-binding protein that is capable of being phosphorylated at T75 by CDK1 and CDK5. Blockage of t-Darpp calcium binding capacity or T75 phosphorylation may therefore help sustain Her2+ breast cancer sensitivity to trastuzumab therapy. Citation Format: Patrycja Magdziarz, You Feng, Dianlu Jiang, Elizabeth Parga, Arianna Celis, Erin Denny, Xiaoying Wang, Martin Phillips, Feimeng Zhou, Susan E. Kane, Jamil Momand. t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5866. doi:10.1158/1538-7445.AM2017-5866

Isolation of a novel calcium-binding peptide from wheat germ protein hydrolysates and the prediction for its mechanism of combination

To isolate a novel peptide with specific calcium-binding capacity, wheat germ protein was hydrolyzed. The hydrolysates were purified using ultrafiltration, anion-exchange chromatography, gel filtration chromatography, and reversed-phase high performance liquid chromatography. The amino acid sequence of the purified peptide was determined and confirmed to be FVDVT (Phe-Val-Asp-Val-Thr). The calcium-binding capacity of FVDVT reached 89.94±0.75%, increased by 86.37% compared to the hydrolysates. The chelating mechanism between FVDVT and calcium was further investigated by Ultraviolet–Visible absorption spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and 1H nuclear magnetic resonances spectroscopy. The results indicated that the oxygen atoms of the carboxy group and the nitrogen atoms of the amido group provided major binding sites. In addition, aspartic acid and threonine show considerable capacity for incorporating with calcium by donating electron pairs. This study provides a feasible approach to isolate calcium-binding peptides and to clarify the possible binding mechanism of calcium and peptide.

Characterization of sea cucumber (stichopus japonicus) ovum hydrolysates: calcium chelation, solubility and absorption into intestinal epithelial cells.

Sea cucumber (Stichopus japonicus) ovum hydrolysates (SCOHs) chelated with calcium were produced to investigate the characteristics of calcium binding and solubility, as well as to study any effects on calcium absorption by human intestinal epithelial cells. The results of the present study show that the calcium-binding capacity of SCOHs depended greatly on the type of proteases. The maximum level of Ca binding (0.38 mmol L-1 ) occurred when trypsin was used, with a peptide yield of 85.7%. Investigation of the possible chelating modes between SCOHs and calcium ions indicated that calcium ions bound to SCOHs primarily via interactions with carboxyl oxygen and amino nitrogen atoms of Glu and Asp and also that the phosphoserine residues might be also responsible for SCOH-calcium chelation. Moreover, SCOH-calcium complexes maintained the solubility of calcium under simulated gastrointestinal digestion, regardless of the presence of dietary components such as oxalate. Furthermore, SCOH-Ca led to higher peak intracellular [Ca2+ ]i in both Caco-2 cells (338.3 nmol L-1 versus 269.6 nmol L-1 ) and HT-29 cells (373.9 nmol L-1 versus 271.7 nmol L-1 ) than casein phosphopeptide-Ca. Carboxyl oxygen and amino nitrogen atoms in the SCOHs could bind calcium ions, forming SCOH-calcium complexes. These complexes improved calcium solubility under simulated gastrointestinal digestion and also promoted calcium absorption in Caco-2 and HT-29 cells. © 2017 Society of Chemical Industry.

Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid.

Egg yolk phosvitin, one of the most highly phosphorylated extracellular matrix proteins known in nature, has a strong calcium binding and reducing capacity. Here, we investigated the effects of phosvitin on osteoblast differentiation and osteogenic gene expression in cultured mouse osteoblastic MC3T3-E1 cells by using alkaline phosphatase activity analysis, alizarin red S staining and real-time PCR assay. Alkaline phosphatase activity and alizarin red S staining analyses demonstrated no significant difference between differentiating MC3T3-E1 cells cultured in the presence of phosvitin and those cultured in the presence of ascorbic acid after 21 days of differentiation. Our real-time PCR assay also indicated the two groups were similar in the expression of the osteogenic gene markers, collagen type I, osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2. Our findings indicate that phosvitin plays a similar role to that of ascorbic acid in osteoblast differentiation and mineralisation. © 2017 Society of Chemical Industry.

Impact of experimental hypercalcemia on routine haemostasis testing.

BackgroundThe blood to anticoagulant ratio is standardized according to the physiological calcium concentration in blood samples conventionally used for hemostasis testing. Specifically, one fixed volume of 0.109 mmol/L sodium citrate is added to 9 volumes of blood. Since little is known about the impact of hypercalcemia on the calcium-binding capacity of citrate, this study was planned to investigate the effect of experimental hypercalcemia on routine hemostasis testing.MethodsFifteen pooled citrated plasmas with matching lithium-heparin pooled plasma from patients with different values of prothrombin time (PT) were divided in three aliquots of 0.6mL each. The first paired aliquots of both citrate and lithium-heparin plasma were supplemented with 60μL of saline, the second paired aliquots with 30μL of saline and 30μL of calcium chloride and the third paired aliquots with 60μL of calcium chloride. Total and ionized calcium was measured in all aliquots of citrate and lithium-heparin plasma, whereas PT, activated partial thromboplastin time (APTT) and fibrinogen were measured in citrate plasma aliquots.ResultsTotal calcium concentration gradually increased in both lithium-heparin and citrate plasma aliquots 2 and 3 compared to baseline aliquot 1. The concentration of ionized calcium also gradually increased in lithium-heparin plasma aliquots 2 and 3, whereas it remained immeasurable (i.e., <0.10 mmol/L) in all citrate plasma aliquots. No significant differences were observed for values of PT, APTT and fibrinogen in citrate plasma aliquots 2 and 3 compared to the baseline aliquot 1, with a mean bias was always comprised within the desirable quality specifications derived from biological variability data.ConclusionHypercalcemia, up to severe hypercalcemia does not generate significant bias in results of first-line coagulations tests, so that hypothetical consideration of adjusting citrate-blood ratio is unjustified in hypercalcemic patients.

A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

Effects of soil salinity on the content, composition, and ion binding capacity of glomalin-related soil protein (GRSP).

Soil aggregation, an ecosystem function correlated with the concentration of glomalin-related soil protein (GRSP), is highly disturbed in saline soil. However, few studies have focused on differences in amount, composition, and ion binding capacity of GRSP in typical sodic-saline soils. In this study, a field study was performed in Songnen Plain. Combined indicators of soil salinity (Q value) were significant negatively correlated with GRSP concentration by Principal Component Analysis. Multiple linear regression models showed that soil salinity might account for 46%, 25% and 44% variation in total GRSP (T-GRSP), easily-extractable GRSP (EE-GRSP) and difficultly-extractable GRSP (DE-GRSP), respectively. Soil bulk density had most important impact on GRSP concentration, followed by the pH, soil EC had the weak influence. Comparative analysis was carried out between low-salinity and high-salinity soil. Purified T-GRSP of high-saline soil contained higher N content (13.13%), lower C content (43.41%) and lower functional groups relative content (e.g. CO and SiOSi). Purified T-GRSP of high-salinity soil had a greater binding capacity with calcium and phosphorus, the binding capacity could compensate the GRSP loss about 29.8% and 14.1%, respectively. Our findings suggested that sodic salinization of the soil led to a decrease in GRSP concentration and a change in the component percentages. This change in composition might be related to adaptation of fungi-plant systems to varied environments. The calcium and phosphorus binding capacity had a positive dependent of soil salinization, which was possible to develop ecological management or recovery technology in the future.

Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells.

Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives.

The complex nature of calcium cation interactions with phospholipid bilayers.

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Molecular basis of a novel renal amyloidosis due to N184K gelsolin variant.

Mutations in gelsolin are responsible for a systemic amyloidosis first described in 1969. Until recently, the disease was associated with two substitutions of the same residue, leading to the loss of the calcium binding site. Novel interest arose in 2014 when the N184K variant of the protein was identified as the etiological agent of a novel kidney-localized amyloidosis. Here we provide a first rationale for N184K pathogenicity. We show that the mutation induces a destabilization of gelsolin second domain, without compromising its calcium binding capacity. X-ray data combined with molecular dynamics simulations demonstrates that the primary source of the destabilization is a loss of connectivity in proximity of the metal. Such rearrangement of the H-bond network does not have a major impact on the overall fold of the domain, nevertheless, it increases the flexibility of a stretch of the protein, which is consequently processed by furin protease. Overall our data suggest that the N184K variant is subjected to the same aberrant proteolytic events responsible for the formation of amyloidogenic fragments in the previously characterized mutants. At the same time our data suggest that a broader number of mutations, unrelated to the metal binding site, can lead to a pathogenic phenotype.

Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues.

BackgroundAggregation of malformed proteins is a key feature of many neurodegenerative diseases, but the mechanisms that drive proteinopathy in the brain are poorly understood. We aimed to characterize aggregated proteins in human brain tissues affected by dementia.ResultsTo characterize amyloidal plaque purified from post-mortem brain tissue of dementia patient, we applied ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC) coupled mass spectrometry-based proteomics technologies. Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins. Amyloidal plaques were enriched in the deamidated variant of protein S100A9, and structural analysis indicated that both the low- and high-affinity calcium binding motifs of S100A9 were deamidated exclusively in the aggregated fraction, suggesting altered charge state and function of this protein in brain tissues affected by dementia. The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca++ binding, suggesting increased capacity to form pathological aggregates in the brain.ConclusionUC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0200-z) contains supplementary material, which is available to authorized users.

Soybean peptide aggregates improved calcium binding capacity

Soybean peptides are believed to have calcium binding sites as Asp and Glu on side chains. However, the effects of peptide aggregation on calcium binding capacity remain unknown. Peptides with different calcium binding capacities (F1 and F2) were separated in this study by calcium immobilized metal affinity chromatography. The calcium binding capacities and apparent molecular weight distributions of F1 and F2 were analyzed before and after the denaturation agents removal. Results showed that F2 with higher calcium binding capacity was more prone to aggregate than F1. The calcium binding capacity of both F1 and F2 decreased after urea or urea and 0.5% SDS treatments. The removal of denaturation agents resulted in peptide reaggregation and recovery of the calcium binding capacity of F2; no significant changes were found for F1. All the findings implied that aggregation of soybean peptides played an important role in calcium binding.

C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition.

BackgroundSkeletal muscle function depends on calcium signaling proteins in the sarcoplasmic reticulum (SR), including the calcium-binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) calcium release channel, and skeletal triadin 95 kDa (trisk95) and junctin, proteins that bind to calsequestrin type 1 (CSQ1) and ryanodine receptor type 1 (RyR1). CSQ1 inhibits RyR1 and communicates store calcium load to RyR1 channels via trisk95 and/or junctin.MethodsIn this manuscript, we test predictions that CSQ1’s acidic C-terminus contains binding sites for trisk95 and junctin, the major calcium binding domain, and that it determines CSQ1’s ability to regulate RyR1 activity.ResultsProgressive alanine substitution of C-terminal acidic residues of CSQ1 caused a parallel reduction in the calcium binding capacity but did not significantly alter CSQ1’s association with trisk95/junctin or influence its inhibition of RyR1 activity. Deletion of the final seven residues in the C-terminus significantly hampered calcium binding, significantly reduced CSQ’s association with trisk95/junctin and decreased its inhibition of RyR1. Deletion of the full C-terminus further reduced calcium binding to CSQ1 altered its association with trisk95 and junctin and abolished its inhibition of RyR1.ConclusionsThe correlation between the number of residues mutated/deleted and binding of calcium, trisk95, and junctin suggests that binding of each depends on diffuse ionic interactions with several C-terminal residues and that these interactions may be required for CSQ1 to maintain normal muscle function.