• 분석과학
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• 제24권6호
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• pp.527-532
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• 2011

IQGAP1은 세포 내에서 암세포화, 세포이동과 같은 다양한 기능을 수행하고 있으며, $Ca^{2+}$-비의존성 calmodulin (CaM) 결합 단백질로 잘 알려져 있다. IQGAP1내에는 IQ부위가 4 번 반복해서 나타나는데, 이 부위가 IQGAP1의 CaM 결합에 중요한 역할을 한다고 알려져 있다. 이전의 연구를 통해 4개의 IQ 부위 모두 $Ca^{2+}$/CaM과의 결합에 관여하고, IQ3와 IQ4는 $Ca^{2+}$이 결합되지 않는 상태의 CaM인 proCaM 결합에 관여 한다고 알려져 있다. 그러나, 이러한 각각의 IQ 부위와의 결합성이 CaM과 직접적인 결합인지, 아니면 다른 단백질이 매개하는 간접적인 결합인지 알려져 있지 않았다. 따라서, 본 연구에서는 IQGAP1의 각각의 IQ 부위와 CaM의 결합성을 직접적으로 알아보기 의해 in vitro에서 조사해 보았다. 그 결과, 흥미롭게도 이전의 결과와는 다르게 4개의 IQ 부위 중에서 IQ3는 의미있는 $Ca^{2+}$-비의존성 CaM결합성이 있음을 알게 되었고, IQ1는 약한 $Ca^{2+}$-의존성 CaM 결합성이 있음을 알게 되었다. 반면에, 다른 IQ 부위들은 CaM과의 결합력이 약하거나 없음을 확인하였다. 또한, 기존의 IQ 부위 이외에 IQ(2.7-3)과 IQ(3.5-4.4) 부위가 의미있는 CaM 결합성이 있음을 확인하게 되었다. 따라서, 본 연구 결과 CaM이 IQGAP1을 기존의 보고와는 다른 방식으로 조절할 수 있을 가능성이 있음을 알게 되었고, 새로운 결합 부위 동정을 통해 IQGAP1과 CaM의 결합이 미치는 생리학적인 의미를 연구할 수 있는 토대를 마련하였다.

• Molecules and Cells
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• 제39권2호
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• pp.149-155
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• 2016

In the heart, excitation-contraction (E-C) coupling is mediated by $Ca^{2+}$ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the $Ca^{2+}$ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich $Ca^{2+}$ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202-231). Second, in vitro binding assays were conducted to examine the $Ca^{2+}$ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped $Ca^{2+}$ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such $Ca^{2+}$ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of $Ca^{2+}$ into SR at intermediate $Ca^{2+}$ concentrations.

• Journal of Plant Biology
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• 제39권3호
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• pp.173-177
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• 1996

A novel calcium binding protein (CaBP) was purified to electrophoretic homogeneity from Dunaliella salina. In the course of purification experiment, this CaBP was identified as a monomer and its molecular weight was about 21 kDand isoelectric point (pI) value was about 4.1 using isoelectrofocusing. This CaBP was able to bind Ca2+ even in the pressence of an excess MgCl2 and KCI both in solution. In the SDS-PAGE, the Ca2+-bound form was slower than the Ca2+-free form in the nondenaturing PAGE. This means that the CaBP undergoes conformational change in the Ca2+-bound condition. Furthermore, UV absorption spectrum and fluorescence intensity of this CaBP was investigated. UV absorption peak was appeared at about 258 nm and decreased somewhat in Ca2+-bound condition. In the measurement of fluorescence, maximum intensity was appeared at 303 nm and decreased in Ca2+-bound state, similarly as UV absorption spectrum. These show distinct changes upon Ca2+-binding, which indicate of structural and/or dynamic changes largely reminiscent of other members of the EF-hand Ca2+-binding protein family.

• Archives of Pharmacal Research
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• 제23권6호
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• pp.633-636
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• 2000

Translationally controlled tumor protein (TCTP), also known as IgE-dependent histamine-releasing factor, is a growth-related tumor protein. Although the primary sequence of rat TCTP does not reveal any recognizable $Ca^{2+}$ -binding motif, previous studies have demonstrated that rat TCTP consisting of 172 amino acids is a $Ca^{2+}$ -binding protein. However. the region of TCTP required for $Ca^{2+}$ interaction has not been mapped to the molecule. Here, we reported that the $Ca^{2+}$ binding region of TCTP which was mapped by using a combination of deletion constructs of rat TCTP and $^{45}Ca^{2+}$-overlay assay. was confined to amino acid residues 81-112. This binding domain did not show any peculiar loop of calcium- binding motif such as CaLB domain and EF hand motif and it seems to be constituted of random coil regions neighboring the a helix. Thus, our data confirm that TCTP is a novel family of $Ca^{2+}$ -binding protein.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.277-286
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• 2020

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

• 약학회지
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• 제28권3호
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• pp.129-138
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• 1984

Specific [$^{3}H$] ouabain binding and $Ca^{2+}$ -uptake were measured to elucidate the role of high affinity [$^{3}H$] ouabain binding site in rat cardiac myocytes which contain 65% of rod cells. High affinity [$^{3}$H] ouabain binding site, which is about 3% of total pump sites, with apparent dissociation constant ($K_{D}$) of $1.1{\times}10^{-7}M$ and maximum binding site concentration (Bmax) of 1.2 pmol/mg protein ($1.754{\times}10^{5}cells$) were identified. At the concentration of $10^{-7}M$ to $10^{-4}M$, ouabain produced concentration dependent increase in $Ca^{2+}$-uptake of myocytes. The effect of ouabain on $Ca^{2+}$-uptake was not effected by membrane depolarization (elevated K+ in incubation medium) or verapamil. These results suggest that in rat ventricular myocytes the ouabain receptor complex to high affinity site may increase Na+ - $Ca^{2+}$ exchange across the sarcolemmal membrane by inhibition of Na+, K+ - ATPase.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.66-66
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• 2003

In skeletal muscle cells, depolarization of the transverse tubules (T-tubules) results in Ca$\^$2+/ release from the sarcoplasmic reticulum (SR), leading to elevated cytoplasmic Ca$\^$2+/ and muscle contraction. This process has been known as excitation-contraction coupling (E-C coupling). Several proteins, such as the ryanodine receptor (RyR), triadin, junctin, and calsequestrin (CSQ), have been identified to be involved in the Ca$\^$2＋/ release process. However, the molecular interactions between the SR proteins have not been resolved. In the present study, the mechanisms of interaction between RyRl and triadin have been studied by in vitro protein binding and $\^$45/Ca$\^$2+/ overlay assays. Our data demonstrate that the intraluminal loop II of RyR1 binds to triadin in Ca$\^$2+/-independent manner. Moreover, we could not find any Ca$\^$2+/ binding sites in the loop II region. GST-pull down assay revealed that a KEKE motif of triadin, which was previously identified as a CSQ binding site (Kobayasi et al.,2000 JBC) was also a binding site for RyR1. Our results suggest that the intraluminal loop II of RyR could participate in the RyR-mediated Ca$\^$2+/ release process by offering a direct binding site to luminal triadin.

• Journal of Plant Biotechnology
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• 제36권1호
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• pp.53-58
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• 2009

Calcium signals can be transduced by binding calmodulin (CaM), a $Ca^{2+}$ sensor in eukaryotes, is known to be involved in the regulation of diverse cellular functions. We isolated a CaM-binding protein 63 kD (AtCBP63) from the pathogen-treated Arabidopsis cDNA expression library. Recently, AtCBP63 was identified as a CaM bining protein. The CaM binding domain of AtCBP63 was reported to be located in its N-terminal region, In this study, however, we showed that ACaM2 could specifically bind to second CaM-binding domain (CaMBD) of AtCBP63 at the C-terminal region. The specific binding of CaM to CaM binding domain was confirmed by a gel mobility shift assay, a split ubiquitin assay, site-directed mutagenesis, and a competition assay using a $Ca^{2+}$/CaM-dependent enzyme. The gene expression of AtCBP63 was induced by pathogens and pathogens related second messengers. This result suggests that a CaM binding protein, AtCBP63, may play role in pathogen defense signaling pathway.

• Parasites, Hosts and Diseases
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• 제53권3호
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• pp.335-339
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• 2015

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of $Ca^{2+}$, $Mg^{2+}$, $K^+$, and $HCO_3{^-}$ in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.182-182
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• 1996

Annexin I is a member of the annexin family of calcium dependent phospholipid binding proteins and has anti-inflammatory activity by inhibiting phospholipase A$_2$ (PLA$_2$). Recent X-ray crystallographic study of annexin I identified six Ca$\^$2+/ binding bites, which was different types (type II, III) from the well-known EF-hand motif (type I). In this work, the structure of annexin I was studied at atomic level by using $^1$H, $\^$15/N and $\^$l3/C NMR(nuclear magnetic resonance) spectroscopy, and the effect of Ca$\^$2+/ binding on the structure of annexin I was studied, and compared with that of Mg$\^$2+/ binding, When Ca$\^$2+/ was added to annexin I, NMR peak change was occured in high- and low-field regions of $^1$H-NMR spectra. NMR peak change by Ca$\^$2+/ binding was different from that by Mg$\^$2+/ binding. Because annexin I is a larger protein with 35 kDa molecular weight, site-specific (amide-$\^$15/N, carbonyl-$\^$l3/C) labeling technique was also used. We were able to detect methionine, tyrosine and phenylalanine peaks respectively in $\^$13/C-NMR spectra, and each residue was able to be assigned by the method of doubly labeling annexin I with [$\^$13/C] carbonyl-amino acid and [$\^$15/N] amide-amino acid. In $\^$l3/C-NMR spectra of [$\^$13/C] carbonyl-Met labeled annexin I, we observed that methionine residues spatially located near Ca$\^$2+/ binding Sites Were Significantly effected by Ca$\^$2+/ binding. From UV spectroscopic data on the effect of Ca$\^$2+/ binding, we knew that Ca$\^$2+/ binding sites of annexin I have cooperativity in Ca$\^$2+/ binding. The interaction of annexin I with PLA$_2$ also could be detected by using heteronuclear NMR spctroscopy. Consequently, we expect that the anti-inflammatory action mechanism of annexin I may be a specific protein-protein interaction. The residues involved in the interaction with PLA$_2$ can be identified as active site by assigning NMR peaks effected by PLA$_2$ binding.