• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1459-1464
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• 2004

The purpose of this research was to investigate the potential use of cheese whey protein (CWP), a cheese by-product. The physiological activity of calcium-binding peptides in CWP may be used as a food additive that prevents bone disorders. This research also examined the characteristics of calcium-binding peptides. After the CWP was heat treated, it was hydrolyzed by trypsin. Then calcium-binding peptides were separated and purified by ion-exchange chromatography and reverse phase HPLC, respectively. To examine the characteristics of the purified calcium-binding peptides, amino acid composition and amino acid sequence were analyzed. Calcium-binding peptides with a small molecular weight of about 1.4 to 3.4 kDa were identified in the fraction that was flowed out from 0.25 M NaCl step gradient by ion-exchange chromatography of tryptic hydrolysates. The results of the amino acid analysis revealed that glutamic acid in a calcium-binding site took up most part of the amino acids including a quantity of proline, leucine and lysine. The amino acid sequence of calcium-binding peptides showed Phe-Leu-Asp-Asp-Asp-Leu-Thr-Asp and Ile-Leu-Asp-Lys from $\alpha$-LA and Ile-Pro-Ala-Val-Phe-Lys and Val-Tyr-Val-Glu-Glu-Leu-Lys from ${\beta}$-LG.

• Biomolecules & Therapeutics
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• v.15 no.3
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• pp.156-161
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• 2007

Rapid advances in pharmacogenomic research have provided important information to improve drug selection, to maximize drug efficacy, and to minimize drug adverse reaction. The SNPs that are the most abundant type of genetic variants have been proven as valid biomarkers to give information on the prediction of pharmacokinetic/pharmacodynamic properties of drugs based on genotype. In order to elucidate a correlation between SNPs of calcium channel encoding gene and adverse reactions of calcium channel blockers, we investigated SNPs in CACNA1C gene known as a binding site of calcium channel blocker. 96 patients with hypertension who had taken or are taking an antihypertensive drug, 1,4-dihydropyridine (DHP) were included for analysis. These patients were composed of 47 patients with adverse drug reactions (ADR) such as edema from calcium channel blockers and 49 patients without ADR as a control group. The exons encoding the drug binding sites were amplified by PCR using specific primers, and SNPs were analyzed by direct sequencing. We found that there was no SNP in the exons encoding DHP binding site, but four novel SNPs in the exon-intron junction region. However, four novel SNPs were not associated with the ADR of calcium channel blockers. In conclusion, this study showed that ADR from calcium channel blockers may not be caused by SNPs of the binding sites of calcium channel blockers in CACNA1C gene.

• BMB Reports
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• v.33 no.5
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• pp.391-395
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• 2000

Calexcitin, a calcium-binding protein, was previously cloned and functionally characterized in the squid Loligo pealei. We now report the cloning of a second form of Calexcitin, Calexcitin-2, found in the squid Todarodes pacificus optic lobe. Calexcitin-2 has a significantly different carboxyl terminal region than Calexcitin-1. It lacks the CAAX motif, which is a farnesylation site. The amino acid sequence of Calexcitin-2 shows an 84% identity with Calexcitin-1 and also displays a strong cross immunoreactivity. Western blotting shows that Calexcitin-2 was expressed exclusively in the optic lobe region of squid, but not in other body organs. Regardless of its lack of conserved regions for GTP-binding, Calexcitin-2 shows moderately low affinity GTP-binding and also shows dramatic conformational change induced by GTP-binding. Three possible GTP-binding region mutations, K142A, D144A, and K157A, did not change the G TP binding affinity. This raises the possibility that Calexcitin-2 may have a novel GTP-binding motif.

• Biomolecules & Therapeutics
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• v.1 no.1
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• pp.1-8
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• 1993

It has been known that calcium antagonists also inhibit the radioligand binding to muscarinic and $\alpha$-adrenergic receptors and, in case of verapamil, these inhibitions may play a role in the effects of verapamil on the heart. In this study, the effects of nicardipine, nifedipine, nimodipine, diltiazem and verapamil on the binding of [$^3H$]dihydroalprenolol (DHA) to dog cardiac ${\beta}$-adrenergic receptors were examined. A single uniform [$^3H$]DHA binding site ($K_D/= 5nM\;and\;B_{max}=2600$ fmol/mg protein) was identified in dog cardiac sarcolemma. [$^3H$]DHA binding was not affected by the usual therapeutic concentrations of these calcium antagonists (nanomolar range) but in the "nonspecific"concentration ranges ($28-180{\mu}m$) these drugs inhibited [$^3H$]DHA binding to $\beta$-adrenergic receptors. Nicardipine, nifedipine, nimodipine and diltiazem competed for [$^3H$]DHA binding to ${\beta}$-adrenergic receptors with dissociation constants ($K_i$) of $28{\mu}m,\' 74{\mu}m, 39{\mu}m \;and \;35{\mu}m,$ respectively. Verapamil ($K_i=176.5 {\mu}m$) was less potent inhibitor than other drugs and this inhibition was noncompetitive; the maximal binding capacity ($B_{max}$) $300 {\mu}m$ verapamil without change in the apparent dissociation constant (4K_D$) for DHA. These results indicate that the inhibitory action of calcium antagonists at high concentrations on ${\beta}$-adrenergic receptors is not involved in the therapeutic effects of these drugs by the calcium channel blocking action.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.86-86
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• 1997

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A$_2$ (PLA$_2$) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I (Δ-annexin I) and its interactions with Ca$\^$2+/, ATP and cAMP were studied at atomic level by using $^1$H, $\^$15/N, $\^$l3/C NMR (nuclear magnetic resonance) spectroscopy. The effect of Ca$\^$2+/ binding on the structure of Δ-annexin I was investigated, and compared with that of Mg$\^$2+/ binding. The addition of Ca$\^$2+/ to Δ-annexin I caused some changes in the high field and low field regions of $^1$H NMR spectra. Whereas, upon addition of Mg$\^$2+/ to Δ-annexin I, almost no change could be observed. Also we found that the binding ratio of ATP to Δ-annexin I is 1. Because Δ-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-$\^$l3/C, amide-$\^$15/N) labeling technique was used to determine the interaction sites of Δ-annexin I with Ca$\^$2+/ and ATP. Assignments of all the histidinyl carbonyl carbon resonances have been completed by using Δ-annexin I along with its specific 1,2-subdomain. The carbonyl carbon resonances originating from His52 and His246 of Δ-annexin I were significantly affected by Ca$\^$2+/ binding, and some Tyr and Phe resonances were also affected. The carbonyl carbon resonances originating from His52 is significantly affected by ATP binding, therefore His52 seems to be involved in the ATP binding site of Δ-annexin I.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.375-377
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• 2000

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the hydrolysis of phosphatidyl-4,5-bisphosphate catalysed by phosphatidylinositol - specific phospholipase C yields to D-myo-inositol - 1,4,5-trisphosphate and diacylglycerol, which are well known second messengers. The binding of InsP$_3$to a receptor located on the endoplasmic reticulum triggers a calcium release from the endoplasmic reticulum. We have detected and partially characterised key components of phosphoinositide signaling. First, tobacco microsomal fraction and plasma membrane PI-PLC. Consecutively, using a radioligand binding assay we have identified a $Ca^{2+}$ -dependent high affinity InsP$_3$binding site in microsomal membrane fraction vesicle preparation and then we have measured inositol-1,4,5-trisphosphate induced calcium release from tobacco microsomal fraction. These findings suggest that phosphoinositide signaling system is present and operates in the tobacco suspension culture.e.

• Korean Journal of Veterinary Research
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• v.48 no.3
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• pp.243-250
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• 2008

A calmodulin-dihydrofolate reductase (DHFR) sandwich fusion protein was generated by insertion of calmodulin into the $\beta$-bulge region of DHFR to observe the effects of structurally constraining the calmodulin structure. The calcium binding properties of the sandwich protein were almost identical to calmodulin. Similar to calmodulin ($10.7 {\mu}M$), the sandwich protein bound four equivalents of calcium, with half saturation ($K_{0.5}$) observed at a [$Ca^{2+}$] of $8{\mu}M$. However, nicotinamide adenine dinucleotide (NAD) kinase activation property of the sandwich protein was lower than that of calmodulin. The sandwich protein activated NAD kinase, but to only half of the level obtained with calmodulin. The K 0.5 for both calmodulin and the sandwich protein were approximately the same (1-2 nM). Methylation analyses of the sandwich protein show that insertion of calmodulin into DHFR results in a large decrease in methylation. The $V_{max}$ observed with the sandwich protein (95 nmole/min/ml) was only 22% of the value observed with calmodulin (436 nmol/min/ml) in the presence of calcium. Addition of trimethoprim to the reaction significantly inhibited the observed methylation rate. Overall, the data suggest that the insertion of calmodulin into the DHFR structure has little effect on calcium binding by the individual lobes of calmodulin, but may constrain the lobes in a manner that results in altered interaction with the calmodulin-dependent proteins, and severely perturbed the methyltransferase recognition site.

• Journal of the Korean Magnetic Resonance Society
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• v.2 no.2
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• pp.141-151
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• 1998

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A2(PLA2) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I ({{{{ DELTA }}-annexin I) and its interactions with Ca2+, ATP and cAMP were studied at atomic level by using nuclear magnetic resonance (NMR) spectroscopy. The effect of Ca2+ binding on the structure of {{{{ DELTA }}-annexin I was investigated. The addition of Ca2+ to {{{{ DELTA }}-annexin I caused some changes in 13C NMR spectra. Carbonyl carbon resonances of some histidines were significantly broadened by Ca2+ binding. However, in the case of methionine, phenylalanine, and tyrosin, small changes could be observed. We found that ATP and cAMP bind {{{{ DELTA }}-annexin I, and the binding ratio of ATP to {{{{ DELTA }}-annexin I is 1. These results are well consistent with the report that cAMP and ATP interact with annexin I, and affect the calcium channels formed by annexin I. Because {{{{ DELTA }}-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-13C) labeling technique was used to study the interaction sites of {{{{ DELTA }}-annexin I with Ca2+. NMR study was focused on the carbonyl carbon resonances of tyrosine, phenylalanine, methionine and histidine residues of {{{{ DELTA }}-annexin I because the number of these amino acids is small in the amino acid sequence of {{{{ DELTA }}-annexin I.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.3
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• pp.219-227
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• 2019

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.57-66
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• 1996

To investigate properties of Ca-release channel in the reptile skeletal muscle, electrophoretical analysis, purification of RyR, $[^3H]ryanodine$binding study, and $^{45}Ca-release$ were carried out in the SR vesicles prepared from the snake skeletal muscle. The snake SR vesicle has the single high molecular weight protein band on SDS-PAGE, and its mobility was similar with that of rat skeletal SR vesicles. The high molecular weight band on SDS-PACE was found in the $[^3H]ryanodine$ peak fractions $(Fr_{5-7})$ obtained from the purification step of the RyR. Maximal binding site and Kd of the snake SR RyR were 6.36 pmole/mg protein and 17.62 nM, respectively. Specific binding of $[^3H]ryanodine$ was significantly increased by calcium and AMP (P<0.05), but not or slightly inhibited by tetracaine, ruthenium red (5.4%), or $MgCl_2$ (21%). $^{45}Ca-release$ from the SR vesicles loaded passively was significantly increased by the low concentration of calcium $(1{\sim}10{\mu}M)$ and AMP (5 mM)(P<0.05), but significantly decreased by the high concentration $(300{\mu}M)$ of calcium, tetracaine (1 mM), ruthenium red $(10{\mu}M)$, and $MgCl_2$ (2 mM)(P <0.05). From the above results, it is suggested that snake SR vesicles also have the RyR showing the similar properties to those of mammalian skeletal RyR with the exceptions of no or slight inhibition of $[^3H]ryanodine-binding$ by tetracaine, ruthenium red, or $MgCl_2$.