• BMB Reports
• /
• 제37권2호
• /
• pp.153-158
• /
• 2004

The acid-labile subunit (ALS) is known to interact with the IGF binding protein (IGFBP) in the presence of insulin-like growth factors (IGFs). Studies, however, indicate that ALS forms a doublet with IGFBP3, independent of IGFs. To characterize the structural domain required for the IGF-free ALS-IGFBP3 interaction, seven recombinant human IGFBP3 mutants were generated: three deletion mutants and four site-specific mutants that had altering N-terminal regions of IGFBP3. ALS and IGFBP3 mRNAs were co-injected into Xenopus oocytes, and their products were cross-linked and immunoprecipitated using antisera against ALS or IGFBP3. Among the deletion mutants, the mutant of D40 (deleted in 11-40th amino acids) exerted no effect in the interaction with ALS, while D60 (${\Delta}11$-60) demonstrated a moderate reduction. D88 (${\Delta}11$-88), however, showed a significant decrease. In the case of site-specific mutants, the mutation that alterated the IGF binding site (codons 56 or 80) exerted a significant reduction in the interaction, whereas codons 72 or 87 showed no significant change in the interaction with ALS. The stability of the ALS-IGFBP3 interaction was analyzed according to a time-dependent mode. Consistent with the binding study, mutants on the IGF binding sites (56 or 80) consistently show a weakness in the ALS-IGFBP3 interaction when compared to the mutants that covered the non-IGF binding sites (72 or 87). This study suggests that the N-terminal of IGFBP3, especially the IGF binding site, plays an important role in interacting with ALS as well as in stabilizing the dual complex, independent of IGFs.

• 약학회지
• /
• 제32권2호
• /
• pp.101-111
• /
• 1988

$[^3H]$ Quinuclidinyl benzilate(QNB) binding assays were performed in the dog ventricular sarcolemma fraction enriched approx. 32-fold in sarcolemma compared to the starting homogenate to elucidate the effect of antihistaminics on cardiac muscarinic receptor. Chlorpheniramine(CHP) inhibited specific binding of $[^3H]$QNB and delayed the equilibrium binding. The rate constants at $37^{\circ}C$ for formation and dissociation of the QNB receptor complex were $0.38{\times}10^9\;M^{-1}$ and $1.6{\times}10^{-2}\;min^{-1}$, respectively. The mean value for the dissociation constant from the pairs of the rate constants was 43. 2 pM and this value was similar to the value(44.8pM) determined from Scatchard analysis. CHP decreased association rate constant, indicating increase in $K_D$ value. Decrease in affinity without affecting the binding site concentration$(B_{max})$ for $[^3H]$QNB binding by CHP was also demonstrated by Scatchard analysis. $K_i$ values for $H_i$-blockers that inhibited specific $[^3H]$QNB binding were $0.02{\sim}4.8{\mu}M$. Cimetidine with $K_i$ value of $230{\mu}M$, however, was ineffective in displacing $[^3H]$QNB binding at concentration of $50{\mu}M$. The Hill coefficient for $H_1$-blockers were about one. The results indicate that $H_1$-antihistaminics inhibit $[^3H]$ QNB binding by interaction with myocardiac muscarinic cholinergic receptor and anticholinergic side effects of these drugs are mainly due to this receptor blocking mechanism.

• BMB Reports
• /
• 제43권6호
• /
• pp.407-412
• /
• 2010

Homeodomain (HD) is a highly conserved DNA-binding domain composed of helix-turn-helix motif. Drosophila Vnd (Ventral nervous system defective) containing HD acts as a regulator to either enhance or suppress gene expression upon binding to its target sequence. In this study, kinetic analysis of Vnd binding to DNA was performed. The result demonstrates that DNA-binding affinity of the recombinant protein containing HD and NK2-specific domain (NK2-SD) was higher than that of the full-length Vnd. To access whether phosphorylation sites within HD and NK2-SD affect the interaction of the protein with the target sequence, alanine substitutions were introduced. The result shows that S631A mutation within NK2-SD does not contribute significantly to the DNA-binding affinity. However, S571A and T600A mutations within HD showed lower affinity for DNA binding. In addition, DNA-binding analysis using embryonic nuclear protein also demonstrates that Vnd interacts with other nuclear proteins, suggesting the existence of Vnd as a complex.

• BMB Reports
• /
• 제29권1호
• /
• pp.1-10
• /
• 1996

The binding interaction of ethidium to a series of synthetic deoxyoligonucleotides containing a B-Z junction between left-handed Z-DNA and right-handed B-DNA, was studied. The series of deoxyoligonucleotides was designed so as to vary a dinucleotide step immediately adjacent to a B-Z junction region. Ethidium binds to the right-handed DNA forms and hybrid B-Z forms which contain a B-Z junction, in a highly cooperative manner. In a series of deoxyoligonucleotides, the binding affinity of ethidium with DNA forms which were initially hybrid B-Z forms shows over an order of magnitude higher than that with any other DNA forms, which were entirely in B-form DNA The cooperativity of binding isotherms were described by an allosteric binding model and by a neighbor exclusion model. The binding data were statistically compared for two models. The conformation of allosterically converted DNA forms under binding with ethidium is found to be different from that of the initial B-form DNA as examined by CD spectra. The ratio of the binding constant was interestingly correlated to the free energy of base unstacking and the conformational conversion of the dinucleotide. The more the base stacking of the dinucleotide is unstable, or the harder the conversion of B to A conformation, the higher the ratio of the binding constant of ethidium with the allosterically converted DNA forms and with the initial B-Z hybrid forms. DNA sequence around a B-Z junction region affects the binding affinity of ethidium. The results in this study demonstrate that ethidium could preferentially interact with unusual DNA structures.

• Journal of Pharmaceutical Investigation
• /
• 제22권2호
• /
• pp.155-161
• /
• 1992

Although glycosylated liposomes have attracted much attention as targeting delivery systems (DDS) of drugs to specific organs which have glycoside receptors, physical instability of liposomes greatly limits their practical application. In this case, proliposomes might be a potential answer to solve this problem. Utilizing the proliposomes as tageting DDS has been a goal of our series of works; we have tried to develop DDS which form liposomes uppon adding water and can deliver drugs to specific target organs/cells such as hepatocytes. In this paper, preparation of glycosylated liposomes and binding of the liposomes with lectin (agglutinin RCA 120) was studied. Asialoletuin (AF) was selected as a model compound which has galactose terminal and is favorable for binding with galactose receptor on the surface of hepatocytes. AF was obtained by splitting the terminal N-acetylneuraminic acid (NANA) of fetuin. Small unilamellar AF-liposomes were prepared by mixing aqueous solution of AF-palmitate with thin film of phosphatidyl choline and cholesterol (30:10 w/w) formed on the innersurface of the round bottomed flask. They were successively extruded through polycarbonate membranes (0.45 mm). Palmitoyl-AF not incorporated into the liposomal bilayer was separated from liposomes by a Sepharose 4B column equilibrated with 10 mM Tris-HCI buffered saline. Lectin (agglutinin RCA 120) was added to the suspension of AF-liposomes and incubated at $37^{\circ}C$ for 2 hr. After centrifugation, the unbound lectin in the supernatant was assayed for protein. The binding of the lectin to AF-liposomes (AF content 2.8 nmole) at $37^{\circ}C$ was linear at least upto 35 mg of lectin indicating high affinity association of the lectin to AF molecules of the liposomes.

• Journal of Microbiology and Biotechnology
• /
• 제33권7호
• /
• pp.964-972
• /
• 2023

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
• /
• pp.132-134
• /
• 2005

The CC chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP-1-induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP-1 induced angiogenesis. Surface Plasmon Resonance measurements demonstrated specific binding of the peptide to MCP-1 but not to the other homologous protein, MCP-3. Flow cytometry revealed that the peptide inhibited the MCP-1 binding to THP-1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP-1 induced angiogenesis. Our observations indicate that the MCP-1-binding peptide exerts its anti-angiogenic effect by interfering with the interaction between MCP-1 and its receptor.

• 한국자기공명학회논문지
• /
• 제2권1호
• /
• pp.50-58
• /
• 1998

The systematic chirality investigations were made on the basic of the fact that zinc-binding tallysomycin (ZnTLMA) could have chiral centers (Zn, NC3, C6) at possible 4-, 5-, and 6-coordination models. Although our NMR data exhibit that the ligation sites are ${\beta}$-aminoalanine, ${\beta}$-hydroxyhistidine, and pyrimidine moiety, all possible coordination modes were tested out to see what kind of chiralities on NC3-C6 are favorable to each coordination mode. Tests were also made that take into account the specific configuration of functional groups, including ${\beta}$-aminoalanine, sugar ring, and ${\beta}$-hydroxyhistidine. Tests were finally extended to zinc-water binding and specific conformational studies by introducing various hydrogen bonding networks associated with the propionamide side chain and the carbamide group of mannose. Results of systematic chirality investigations exhibit that the S-S configuration of NC3-C6 is favorable to all of coordination models, but the R-S configuration, if exists at all, should have internal strain on C6 chiral center.

• Molecules and Cells
• /
• 제21권3호
• /
• pp.376-380
• /
• 2006

Gene expression is regulated in large part at the level of transcription under the control of sequence-specific transcriptional regulatory proteins. Therefore, the ability to affect gene expression at will using sequencespecific artificial transcription factors would provide researchers with a powerful tool for biotechnology research and drug discovery. Previously, we isolated 56 novel sequence-specific DNA-binding domains from the human genome by in vivo selection. We hypothesized that these domains might be more useful for regulating gene expression in higher eukaryotic cells than those selected in vitro using phage display. However, an unpredictable factor, termed the "context effect", is associated with the construction of novel zinc finger transcription factors--- DNA-binding proteins that bind specifically to 9-base pair target sequences. In this study, we directly selected active artificial zinc finger proteins from a zinc finger protein library. Direct in vivo selection of constituents of a zinc finger protein library may be an efficient method for isolating multi-finger DNA binding proteins while avoiding the context effect.

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 1996년도 정기총회 및 학술발표회
• /
• pp.19-19
• /
• 1996

Cyclic AMP receptor protein (CRP) from E. Coli plays a key role in regulation of the expression of more than 20 genes of the bacterium. CRP binds in the presence of cAMP to a specific target site near the promoter of each gene under its regulation. CRP is a dimer (Mr～47,000) of two identical subunits. There are two binding domains in the CRP monomer, one for the binding of the cAMP and the other for the binding of specific DNA sequences. (omitted)