• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.44-44
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• 2003

$BK_{Ca}$ channels were suggested to contain one or more domains of the ‘regulator of K+ conductance’(RCK) in their cytosolic carboxyl termini (Jiang et al.2001). It was also shown that the RCK domain in mammalian $BK_{Ca}$ channels might sense the intracellular $Ca^{2+}$ with a low affinity (Xia et al. 2002). We aligned the amino acid sequence of the $\alpha$-subunit of rat $BK_{Ca}$ channels (rSlo) with known RCK domains and identified a second region exhibiting about 50％ homology. This putative domain, RCK2, contains the characteristic amino acids conserved in other RCK domains. We wondered whether this second domain is involved in the domain-domain interaction and the gating response to intracellular $Ca^{2+}$ for rSlo channel, as revealed in the structure of RCK domain of E. coli channel (Jiang et al.2001). In order to examine the possibility, site-directed mutations were introduced into the RCK2 domain of rSlo channel and the mutant channels were expressed in Xenopus oocytes for functional studies. One of such mutation, G772D, in the putative nucleotide-binding domain resulted in the enhanced $Ca^{2+}$ sensitivity and the channel gating of rSlo channel. These results suggest that this region of $BK_{Ca}$ channels is important for the channel gating and may form an independent domain in the cytosolic region of $BK_{Ca}$ channels. In order to obtain the mechanistic insights of these results, G772 residue was randomly mutagenized by site-directed mutagenesis and total 17 different mutant channels were constructed. We are currently investigating these mutant channels by electrophysiological techniques.ical techniques.

• Journal of Applied Biological Chemistry
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• v.53 no.3
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• pp.174-178
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• 2010

Calcium and iron binding peptides were prepared by enzymatic hydrolysis and ultrafiltration of rice bran protein (RBP), which was isolated from defatted rice bran by phytase and xylanase treatment and ultrasonication. The isolated RBP had a molecular weight in the range of 10-66 kDa. The extracted proteins were hydrolyzed using Flavourzyme for 6 hr. After ultrafiltration under 5 kDa as molecular weight, the peptides were fractionated into 4 peaks by Sephadex G-15 gel permeation chromatography, and each fraction was determined for calcium and iron binding activity. As the result, Fl and F2 fractions were the best candidate for calcium and iron chelation, respectively. These results suggest that the calcium and iron binding peptides can be used as functional food additives in food industry.

• Journal of Life Science
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• v.19 no.1
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• pp.129-137
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• 2009

Calcium ($Ca^{2+}$) plays pivotal roles as an intracellular second messenger in response to a variety of stimuli, including light, abiotic. and biotic stresses and hormones. $Ca^{2+}$ sensor is $Ca^{2+}$-binding protein known to function in transducing signals by activating specific targets and pathways. Among $Ca^{2+}$-binding proteins, calmodulin (CaM) has been well reported to regulate the activity of down-stream target proteins in plants and animals. Especially plants possess multiple CaM genes and many CaM target proteins, including unique protein kinases and transcription factors. Thus, plants are possible to perceive different signals from their surroundings and adapt to the changing environment. However, the function of most of CaM or CaM-related proteins have been remained uncharacterized and unknown. Hence, a better understanding of the function of these proteins will help in deciphering their roles in plant growth, development and response to environmental stimuli. This review focuses on $Ca^{2+}$-CaM messenger system, CaM-associated proteins and their role in responses to external stimuli of both abiotic and biotic stresses in plants.

• Journal of Life Science
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• v.21 no.5
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• pp.671-677
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• 2011

We previously reported the isolation and characterization of a gene, SCA1 (for soybean $Ca^{2+}$-ATPase 1), encoding a calmodulin-regulated $Ca^{2+}$-ATPase that is located in the plasma membrane in soybean. Here, a $Ca^{2+}$-ATPase designated as SCA2 was isolated from soybean. The two $Ca^{2+}$-ATPases, SCA1 and SCA2, share a remarkably high degree of similarity (78%). Ten transmemebrane domains were predicted by hydropathy analysis. Using gel overlay assays, CaM was found to bind to SCA2 in a $Ca^{2+}$-dependent manner. Southern blot analysis revealed the presence of two copies of the $Ca^{2+}$-ATPase gene in the soybean genome. An N-terminal truncation mutant that deletes sequence through the putative calmodulin binding site was able to complement a yeast mutant (K616) that was deficient in two endogenous $Ca^{2+}$ pumps. Our results indicate that SCA2 is structurally highly conserved with type IIB $Ca^{2+}$ pumps in plants.

• The Korean Journal of Pharmacology
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• v.30 no.1
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• pp.125-136
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• 1994

To characterize the SR Ca-release channel protein complex of crustacean, $^{45}Ca-release,\;[^3H]ryanodine$ binding, and immunoblot studies were carried out in the crayfish and/or lobster skeletal sarcoplasmic reticulum. Bmax and affinity of crayfish SR to ryanodine were lower than those of lobster SR. AMP (5mM) increased $[^3H]ryanodine$ binding significantly in both vesicles (P<0.05). $Mg^{2+}$(5mM) or tetracaine(1mM) inhibited $[^3H]ryanodine$ binding significantly in both vesicles (P<0.001), but ruthenium red $(10\;{\mu}M)$ inhibited it moderately. In SDS polyacrylamide gel electrophoretic analysis of crayfish SR vesicles, there was a high molecular weight band that showed similar mobility with Ca-release channel protein of lobster skeletal SR, but more rapid mobility (HMWBr) than that of rabbit skeletal SR (HMWBS). Immunoblot analysis showed that polyclonal Ab to lobster skeletal SR Ca-release channel protein was react with HMWBr of crayfish skeletal SR, but not with that of HMWBs of rabbit skeletal SR. ^{45}Ca-release from crayfish skeletal SR vesicles was increased by the increase of extravesicular calcium from $1{\mu}M$ to 1mM. This Ca-release phenomenon was similar, but more sensitive in the low concentration of $Ca^{2+}$, compared to that from lobster SR vesicles. AMP (5mM) or caffeine (10mM) did not affect to $^{45}Ca-release.\;^{45}Ca-release$ was inhibited slightly ($3{\sim}8%\;by\;Mg^{2+}$) (5mM) or tetracaine (1mM), and moderately (23%) by high concentration of ruthenium red $(300\;{\mu}M)$. From the above results, it is suggested that SR Ca-release channel protein of crustacean has different properties from that of the rabbit, and similar properties between crayfish and lobster in functional and immunological aspects, but Ca-release via crayfish channel may be more sensitive to calcium.

• Molecules and Cells
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• v.37 no.9
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• pp.656-663
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• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• BMB Reports
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• v.34 no.6
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• pp.573-577
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• 2001

The mammalian heart is known to undergo significant mechanical changes during fetal and neonatal development. The objective of this study was to define the ontogeny of the ryanodine receptor/$Ca^{2+}$ release channel and SERCA that play the major roles in excitation-contraction coupling. Whole ventricular homogenates of fetal (F) (19 and 22 days in gestation), postnatal (N) (1 and 7 days postnatal), and adult (A) (5 weeks postnatal) Sprague-Dawley rat hearts were used to study [$^3H$]ryanodine binding and oxalate-supported $^{45}Ca^{2+}$ uptake. For the ryanodine receptor, the major findings were: (1) The ryanodine receptor density, as determined by maximal [$^3H$]ryanodine binding ($B_{max}$), increased 3 fold between the F22 and A periods ($0.26{\pm}0.1$ vs. $0.73{\pm}0.07$ pmoles/mg protein, p<0.01), whereas there was no significant change during the F22 and N1 development phases ($0.26{\pm}0.1$ vs. $0.34{\pm}0.01$). (2) Affinity of the ryanodine receptor to ryanodine did not significantly change, as suggested by the lack of change in the $K_d$ during the development and maturation. For SERCA, changes started early with an increased rate of $Ca^{2+}$ uptake in the fetal periods (F19: $8.1{\pm}1.1$ vs. F22: $19.3{\pm}2.2$ nmoles/g protein/min; p<0.05) and peaked by 7 days (N7) of the postnatal age ($34.9{\pm}2.1$). Thus, we conclude that the quantitative changes occur in the ryanodine receptor during myocardial development. Also, the maturation of the $Ca^{2+}$ uptake appears to start earlier than that of the $Ca^{2+}$ release.

• Molecules and Cells
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• v.20 no.1
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• pp.51-56
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• 2005

Excitation-contraction coupling (ECC) proteins in the human heart were characterized using human atrial tissues from different age groups. The samples were classified into one infant group (Group A: 0.2-7 years old) and three adult groups (Group B: 21-30; Group C: 41-49; Group D: 60-66). Whole homogenates (WH) of atrial tissues were assayed for ligand binding, $^{45}Ca^{2+}$ uptake and content of ECC proteins by Western blotting. Equilibrium [$^3H$]ryanodine binding to characterize the ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR) showed that the maximal [$^3H$]ryanodine binding ($B_{max}$) to RyR was similar in all the age groups, but the dissociation constant ($k_d$) of ryanodine was higher in the infant group than the adult groups. Oxalate-supported $^{45}Ca^{2+}$ uptake into the SR, a function of the SR SERCA2a activity, was lower in the infant group than in the adult groups. Similarly, [$^3H$]PN200-110 binding, an index of dihydropyridine receptor (DHPR) density, was lower in the infant group. Expression of calsequestrin and triadin assessed by Western blotting was similar in the infant and adult groups, but junctin expression was considerably higher in the adult groups. These differences in key ECC proteins could underlie the different $Ca^{2+}$ handling properties and contractility of infant hearts.

• Journal of Plant Biology
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• v.8 no.1_2
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• pp.19-23
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• 1965

Interaction occuring among the different Ca concentrations, pH and temperatures in the promotive effect of Ca in pollen growth was studied by using pollen from Crinum asiaticum and Cryptostegia grandiflora. Data for pollen tube elongation were found to be more indicative of representing the promotive action of Ca ion in pollen growth than those for pollen germination, and were served to evaluate the experimental results. The pollen growth increased as the concentration of Ca increase. The optimal pH range for pollen growth shifted from the lower pH to the higher as the concentration of Ca increase. The characteristic Ca effect was disappeared, and no pH effect at various ranges was observed when pollen grains were grown at the low temperature(8$^{\circ}C$). The Ca effect became quite pronounced if temperature were raised. The Ca effect became even mroe striking if the condition was in higher pH ranges (weak alkaline). Higher pH ranges were found to be more favorable for the Ca action, whereas higher temperature was required to bring about more pronounced Ca effect. Thus, the longest pollen tube was obtained with the highest pH, temperature adopted for the medium supplemented with Ca in the present experiment, and the shortest tube with the lowest temperature applied at the highest pH. Pectin synthesis in pollen tube was considered as a metabolic process, whereas Ca binding in pectin of the pollen tube wall as non-metabolic in nature. Disappearance of Ca effect at the low temperature was probably brought about by blocking the metabolic synthesis of pectin, and nonmetabolic Ca binding seems to take place more extensively with higher concentrations of Ca and at higher pH levels than the lower.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.1-6
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• 1994

We have studied the relation between Ca$^{2+}$-ATPase and trigger peptidase(TPase) which are membeane protein well known as their significant role for signal transduction of mating pheromone in heterobasidiomycetous yeast. Rhodosporidium toruloides. We found out that there were Ca $^{2+}$-ATPase and TPase together in isolated calmodulim binding protein(CBP), usion calmodulin affinity column chromatography after solubilization of mation type a cell membrane protein, and that the dependence of enzyme activity of both the enzymes on Ca$^{2+}$, phospholipid and nonionic detergent are similar. However, Ca$^{2+}$-ATPase hed quite absolute dependence on calmodulin and, on the other hand, TPase didn't have any dependence. Judging from the fact that there are both enzymes in CBP which the dependence of calmodulin are quite different, we found out that both enzymes were made to their compound and existed in mating type a cell membrane.