• Toxicological Research
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• 제35권1호
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• pp.37-44
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• 2019

A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor ($CB_1$) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified $CB_1$ were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid ${\Delta}^9$-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to $CB_1$ were determined to be (from highest to lowest) $9.52{\times}10^{-3}M$ (JWH-210), $6.54{\times}10^{-12}M$ (JWH-250), $1.56{\times}10^{-11}M$ (${\Delta}^9$-tetrahydrocannabinol), $2.75{\times}10^{-11}M$ (RCS-4), and $6.80{\times}10^{-11}M$ (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.87-87
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• 1995

The muscarinic effects of carbachol were compared on the isolated ileums of guinea-pig, rat and rabbit to elucidate the underlying mechanism of species differences in sensitivity for carbachol. The ED$\_$50/ value estimated on the guinea-pig ileum was 4 to 6-fold lower than those obtained on the rat and rabbit ileums, but the K$\_$A/ values of carbachol determined by functional assays were almost identical with 12-l7 ${\mu}$M in all of three ileums. The competition data of carbachol for [$^3$H]QNB binding were best described by a two-site model yielding the Ki values of 0.4-0.6${\mu}$M and 12-16${\mu}$M for high(K$\_$H/) and low(K$\_$L/) affinity sites, respectively. The low affinity dissociation constants(K$\_$L/) of carbachol determined from receptor binding studies thus were not significantly different from the K$\_$A/ values estimated from functional studies. The percentage of receptor occupation that carbachol requires for half-maximal response was approximately 3 to 5-fold lower in guinea-pig compared to rat and rabbit whereas the density of muscarinic binding sites per gram of ileum measured by [$^3$H]QNB saturation isotherms was two-fold higher in guinea-pig than that in rat and rabbit. Therefore, the numbers of muscarinic receptors occupied at ED$\_$50/ values of carbachol were about two-fold lower in guinea-pig, suggesting two-fold greater intrinsic efficacy. These results indicate that the guinea-pig ileum has higher muscarinic receptor density and greater intrinsic efficacy for carbachol than the rat and rabbit ileums.

• 한국식품과학회지
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• 제30권1호
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• pp.218-223
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• 1998

우유 casein단백질을 alcalase로 가수분해시켰을 때, 생성되는 peptide 중 철분과 결합력이 있는 peptide인 IBP가 $immobilized\;Fe^{3+}\;affinity\;chromatography$에 의하여 용이하게 분리되었다. IBP의 분자량은 2,175 dalton이었으며 $pH\;6,\;37^{\circ}C$에서 1시간동안 일정량의 철분과 항온처리하였을 경우, 25, 50, 100 g/mL의 IBP는 각각 4.2, 5.7, 7.1 g의 철분을 가용화시키는 능력을 보였다. IBP는 proline (24.5 Mol%), lysine (15.7 Mol%), glutamic 또는 glutamine (14.9 Mol%) 등의 아미노산으로 구성되어 있었으며 이들의 N-terminal sequence는Met-Ala-Pro-Lys-His의 순으로 이루어져 있었다. 분자량, 아미산 조성, N-terminal sequence등의 결과를 종합해 보면 IBP는 casein 중 -casein의 아미노산 서열 $102{\sim}119$에 해당하는 18개의 아미노산으로 구성된 peptide임을 알 수 있었다.

• Genomics & Informatics
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• 제13권2호
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• pp.45-52
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• 2015

Acute myeloid leukemia is a well characterized blood cancer in which the unnatural growth of immature white blood cell takes place, where several genes transcription is regulated by the micro RNAs (miRNAs). Argonaute (AGO) protein is a protein family that binds to the miRNAs and mRNA complex where a strong binding affinity is crucial for its RNA silencing function. By understanding pattern recognition between the miRNAs-mRNA complex and its binding affinity with AGO protein, one can decipher the regulation of a particular gene and develop suitable siRNA for the same in disease condition. In the current work, HOXA9 gene has been selected from literature, whose deregulation is well-established in acute myeloid leukemia. Four miRNAs (mir-145, mir-126, let-7a, and mir-196b) have been selected to target mRNA of HOXA9 (NCBI accession No. NM_152739.3). The binding interaction between mRNAs and mRNA of HOXA9 gene was studied computationally. From result, it was observed mir-145 has highest affinity for HOXA9 gene. Furthermore, the interaction between miRNAs-mRNA duplex of all chosen miRNAs are docked with AGO protein (PDB ID: 3F73, chain A) to study their interaction at molecular level through an in silico approach. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding of AGO-assisted miRNA based gene silencing mechanism in HOXA9 gene associated in acute myeloid leukemia computationally.

• Genomics & Informatics
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• 제21권3호
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• pp.32.1-32.11
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• 2023

Resistance to anti-tuberculosis drugs, especially ethambutol (EMB), has been widely reported worldwide. EMB resistance is caused by mutations in the embB gene, which encodes the arabinosyl transferase enzyme. This study aimed to detect mutations in the embB gene of Mycobacterium tuberculosis from Papua and to evaluate their impact on the effectiveness of EMB. We analyzed 20 samples of M. tuberculosis culture that had undergone whole-genome sequencing, of which 19 samples were of sufficient quality for further bioinformatics analysis. Mutation analysis was performed using TBProfiler, which identified M306L, M306V, D1024N, and E378A mutations. In sample TB035, the M306L mutation was present along with E378A. The binding affinity of EMB to arabinosyl transferase was calculated using AutoDock Vina. The molecular docking results revealed that all mutants demonstrated an increased binding affinity to EMB compared to the native protein (-0.948 kcal/mol). The presence of the M306L mutation, when coexisting with E378A, resulted in a slight increase in binding affinity compared to the M306L mutation alone. The molecular dynamics simulation results indicated that the M306L, M306L + E378A, M306V, and E378A mutants decreased protein stability. Conversely, the D1024N mutant exhibited stability comparable to the native protein. In conclusion, this study suggests that the M306L, M306L + E378A, M306V, and E378A mutations may contribute to EMB resistance, while the D1024N mutation may be consistent with continued susceptibility to EMB.

• 대한약리학회지
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• 제27권1호
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• pp.33-43
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• 1991

고혈압백서(1-kidney, 1-clip-hypertensive rat)의 적혈구에서 노화 과정에 따른 Na, K-ATPase의 변동을 관찰하고저 노화적혈구를 분리한다음 세포막에서의 Na-pump 활성도 및 ouabain의 결합실험과 Rb의 세포내 유입실험을 시행하여 다음과 같은 결과를 얻었다. 1. 본 실험에 사용한 고혈압 백서의 혈압은 수축기 및 이완기 혈압이 165.5/119.0 mmHg로 유의하게 증가 하였다. 노화 적혈구의 평균용적(MCV)과 세포막 단백질 함량은 감소되고 혈색소치는 증가되었다. 2. 110 mM NaCl 및 10 mM KCI 존재하에서의 적혈구 세포막 Na, K-ATPase활성도는 대조군에 비해 고혈압군에서 억제 되었으며 양군 모두에서 노화에 의해 그활성도가 감소되었다. 3. 4 mM RbCl존재하에서 Ouabain에 의해 억제되는 Rb의 유입은 정상 및 고혈압군의 노화적혈구에서 약간 감소되었으며 고혈압군의 young erythrocyte에서는 오히려 약간 증가 되었다. 4. 16 mM RbCl 존재하에서 Ouabain에 의해 억제되는 Rb의 유입은 양군의 노화 적혈구에 서는 각군의 young erythrocyte에 비해 약 30-50% 감소되었으며, 고혈압군에서는 특히 young erythrocyte에서 정상군의 young erythrocyte에 비해 유의하게 감소되었다. 5. $0.13{\times}10^{-6}M$과 $1{\times}10^{-6}M$에서의 ouabain binding은 정상군의 노화적혈구에서는 young erythrocyte에 비해 약간 감소되었으나 고혈압군의 노화적혈구에서는 유의하게 감소되었다. 6. $6{\times}10^-6}M$과 $64{\times}10^-6M$ 에서의 ouabain binding은 양군의 노화 적혈구에서는 약간 감소되었지만 유의성은 없었으며 고혈압군의 young erythrocyte 및 노화적혈구에서는 정상군의 young erythrocyte및 노화 적혈구에 비해 유의하게 감소되었다. 이상의 결과로부터 (1) 고혈압쥐의 young erythrocyte에서는 low affinity의 Na-pump수의 감소및 molecular activity의 증가, (2) 정상쥐의 노화 적혈구에서는 molecular activity의 저하, (3) 고혈압쥐의 노화적혈구에서는 molecular activity의 저하 및 high affinity와 low affinity의 Na-pump수의 저하등에 의하여 Na-pump의 기능이 변동될 수 있을 것으로 추측된다.

• 산업기술연구
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• 제36권
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• pp.33-37
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• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• BMB Reports
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• 제31권5호
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• pp.498-502
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• 1998

HBV polymerase shares several regions of amino acid homology with other DNA-directed and RNA-directed polymerases. The amino acid residues $Asp^{429}$, $Gly^{518}$, $Asp^{551}$, $Lys^{585}$, and $Gly^{641}$ in the conserved motifs A, B', C, D, and E in the polymerase domain of HBV polymerase were mutated to alanine or histidine by in vitro site-directed mutagenesis. Those mutants were overexpressed, purified, and analyzed against DNA-dependent DNA polymerase activity and affinity for DNA binding. All those mutants did not show DNA-dependent DNA polymerase activities indicating that those five amino acid residues are all critical in DNA polymerase activity. South-Western analysis shows that amino acid residues $ASp^{429}$ and $ASp^{551}$ are essential to DNA binding, and $Gly^{318}$ and $Gly^{585}$ also affect DNA binding to a certain extent.

• BMB Reports
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• 제31권5호
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• pp.427-431
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• 1998

In the previous report (Choi et al., 1997), the angiogenin binding peptides identified from a phage-peptide library were analyzed by using the fusion proteins composed of the Escherichia coli maltose binding protein and its corresponding peptides. However, it was difficult to obtain a sufficient amount of the fusion proteins required for further analysis because of the low expression level. We now report a high level expression of the fusion protein and analysis of its anti-angiogenin activity. The use of strong T7 promoter and removal of signal sequence allowed about a 20-fold increase in the expression efficiency of the fusion protein. We were able to obtain about 10 mg of purified fusion protein from one liter of culture. The purified fusion protein showed angiogenin-specific affinity and inhibited the binding of biotinylated actin to human angiogenin at $IC_{50}$ of 0.6 mM. Its anti-angiogenin activity was also revealed by the chorioallantoic membrane assay.