• BMB Reports
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• v.29 no.4
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• pp.353-358
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• 1996

Bovine angiogenin (bAng) is a potent blood vessel inducing protein purified from cow In ilk. fluorescence spectroscopy has been used to study the interaction of bAng with actin in 50 mM Tris-HCl pH 7.5, and 1 mM $CaCl_2$ at $25^{\circ}C$. Actin contains four tryptophans but bAng contains no tryptophans. A 50% decrease in intrinsic fluorescence accompanied formation of the bAng/actin complex. By contrast, the interaction of RNase A, a homologous protein to bAng, with actin results in about 10% quenching of the fluorescence. Fluorescence titration experiments were performed by adding increasing concentrations of bAng (0~1.0 ${\mu}M$) to a constant concentration of actin (0.1 ${\mu}M$), and the dissociation constant $K_d$ for the bAng/actin complex and the stoichiometry n were measured as $20{\pm}1$ nM and $1.0{\pm}0.1$ respectively. These results suggest that the interaction between bAng with actin is specific and that quenching of actin fluorescence has occurred in the bAng/actin complex. The bAng binding sites of actin are discussed in the results of this study, and we propose that Trp-80 in the small domain of bovine actin is responsible for the bAng/actin binding.

• Current Research on Agriculture and Life Sciences
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• v.32 no.3
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• pp.149-154
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• 2014

It is already known that adzuki beans (Vigna angularis) are able to control appetite. Therefore, this study tested the proteins isolated from adzuki beans for their protease resistance and interaction with the intestinal mucosa. The major proteins from adzuki beans were found to be resistant to the digestive enzymes pepsin and pancreatin, and were identified using 2D-SDS-polyacrylamide gel electrophoresis and mass spectrometry. The major adzuki proteins were easily fractionated by treating the soluble protein extract with 10mM $CaCl_2$, and were found to contain lactotransferrin, a homologous protein to the dynein light chain domain, proteinase inhibitor, and proteins with unknown functions. From a tissue binding assay using mouse intestinal tissue sections, the major protein fraction showed weak, yet significant and specific binding to the mucosa layer of the small intestine. Thus, the current results suggest that adzuki proteins are resistant to digestive enzymes, which enables them to survive protease digestion in the intestinal tract, plus they may interact with the intestinal mucosa layer. Therefore, the molecules responsible for controlling appetite in adzuki beans are presumably protease-resistant proteins that interact with the intestinal mucosa or delay digestion in the digestive tract.

• The Korean Journal of Pharmacology
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• v.31 no.2
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• pp.207-217
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• 1995

To investigate the functional and immunological properties of the Ca-release channel in the sarcoplasmic reticulum(SR) of the eel skeletal muscle, $[^3H]ryanodine$ binding, SDS gel electrophoresis, $^{45}Ca\;release$ studies, and immunoblot assay were carried out in the SR of the eel skeletal muscle. Maximal binding sites(Bmax) and $K_D$ values of $[^3H]ryanodine$ for Ca-release channel of the SR of the eel skeletal muscle were $19.44{\pm}1.40\;pmole/mg$ protein and $15.55{\pm}1.69\;nM$, respectively. $[^3H]Ryanodine$ binding to RyR was increased by calcium and AMP. The SR of the eel skeletal muscle has two high molecular weight bands on the SDS PAGE. The mobility of upper band was more slower than the single band of the rabbit skeletal muscle, and that of the lower band was similar with the single band of canine cardiac muscle. Vesicular $^{45}Ca-release$ was activated by calcium. Ca-induced $^{45}Ca-release$ was significantly inhibited by $MgCl_2(2\;mM)$, ruthenium red$(10\;{/mu}M)$ or tetracaine(1 mM), but not by high concentration of calcium itself. AMP-induced $^{45}Ca-release$ was slightly occurred only in the absence of calcium, it was not inhibited by $MgCl_2$ or ruthenium red. Caffeine also increased $^{45}Ca-release$ from the SR vesicles, but it was not affected by $MgCl_2$ or ruthenium red. Polyclonal Ab against rat skeletal muscle RyR is reacted with that of rabbit, but not reacted with that of the eel skeletal muscle. These results suggested that ryanodine receptor of the SR of the eel skeletal muscle is showing some similar properties with that of mammalian skeletal muscle, but might be an another isotype channel having two bands which is less sensitive to AMP, not cross-reacted with antisera against rat RyR, and not inhibited by high concentration of calcium.

• Journal of Life Science
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• v.23 no.3
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• pp.347-354
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• 2013

Innate immunity is the ability to differentiate infectious agents from self. The innate immune system is comprised of a complicated network of recognition and effector molecules that act together to protect the host in the early stage of an infectious challenge. Mannose-binding lectin (MBL or mannose-binding protein, MBP) belongs to the family of $Ca^{2+}$-dependent lectins (C-type lectin with a collagen-like domain), which are considered an important component of innate immunity. While it is associated with increased risk and severity of infections and autoimmunity, the most frequent immuno-deficiency syndrome was reported to be low MBL level in blood. Deficiency of human MBL is caused by mutations in the coding region of the MBL gene. Rat homologue gene of human MBL gene was used to study functions of wild type and mutant MBL proteins. Although extensive studies have yielded the structural information of MBL, the functions of MBL, especially mutant MBL, still require investigation. We previously reported the cloning of rat wild-type MBL gene and the production of a truncated form of MBL protein and its antibody. Here, we present the cloning of mutant MBL cDNA in collagen-like domain (R40C, G42D, and G45E) using site-directed mutagenesis and differential behaviors of wild type and mutant MBL in cells. The major difference between wild type and mutant MBL was that while wild type MBL was secreted, mutant MBL was inhibited for secretion, retained in endoplasmic reticulum, and still functioned as a lectin.

• Molecules and Cells
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• v.27 no.3
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• pp.307-312
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• 2009

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a $Ca^{2+}$-free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by $GDP-{\beta}-S$, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external $Ca^{2+}$ influx, phospholipase C activation, and $Ca^{2+}$ release from internal storage in a G protein-independent and protein kinase C-independent manner.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.10a
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• pp.105-105
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• 2003

• Journal of Photoscience
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• v.9 no.2
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• pp.281-283
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• 2002

S-modulin in frog or its bovine homologue, recoverin, is a 26 kDa EF-hand $Ca^{2+}$-binding protein found in rod photoreceptors. The $Ca^{2+}$ -bound form of S-modulin binds to rhodopsin kinase (Rk) and inhibits its activity. Through this regulation, S-modulin is believed to modulate the light-sensitivity of a rod. In the present study, we tried to identify the interaction site of the $Ca^{2+}$ -bound form of S-modulin to Rk. First, we mapped roughly the interaction regions by using partial peptides of S-modulin. The result suggested that a specific region near the amino terminus is the interaction site of S- modulin. We then identified the essential amino acid residues in this region by using S-modulin mutant proteins: four amino acid residues were suggested to interact with Rk. These residues are located in a small closed pocket in the $Ca^{2+}$-free, inactive form of S-modulin, but exposed to the surface of the molecules in the $Ca^{2+}$ -bound, active form of S-modulin. Two additional amino acid residues were found to be crucial for the $Ca^{2+}$ -dependent conformational changes of S-modulin. The present study firstly identified the functional site of S-modulin, a member of a neuronal calcium sensor protein family.in family..

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• Molecules and Cells
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• v.43 no.11
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• Bulletin of the Korean Chemical Society
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• v.25 no.7
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• pp.1031-1035
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• 2004

The adsorption kinetics of ${\beta}$-casein on a hydrophobic surface has been studied by means of the quartz crystal microbalance (QCM). The self assembled monolayer of 1-octadecanethiol on a gold coated quartz crystal was used as a hydrophobic surface for adsorption. The adsorption kinetics was monitored in different solution conditions. Formation of monolayer is observed in most cases. At high concentration of protein, micelle formation which is interrupted by high ionic strength of solution is observed. Casein binding cations such as $Ca^{2+},\;Ba^{2+}\;and\;Al^{3+}$ increase the hydrophobicity of the protein and the multiple layer adsorption occurs. The strong and weak points of the QCM method in the study of protein adsorption are discussed.