• Biomolecules & Therapeutics
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• v.30 no.4
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• pp.328-333
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• 2022

Repeated morphine administration induces tolerance to its analgesic effects. A previous study reported that repeated morphine treatment activates transient receptor potential vanilloid type 1 (TRPV1) expression in the sciatic nerve, dorsal root ganglion, and spinal cord, contributing to morphine tolerance. In the present study, we analyzed TRPV1 expression and binding sites in supraspinal pain pathways in morphine-tolerant mice. The TRPV1 mRNA levels and binding sites were remarkably increased in the cortex and thalamus of these animals. Our data provide additional insights into the effects of morphine on TRPV1 in the brain and suggest that changes in the expression of, and binding to TRPV1 in the brain are involved in morphine tolerance.

• YAKHAK HOEJI
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• v.36 no.3
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• pp.220-229
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• 1992

The muscarinic acetylcholine receptors of the dog unpregant uterus were characterized using $[^3H]quinuclidinyl$ benzilate(QNB) as a radioligand and the binding of muscarinic receptor agonists and antagonists in the uterus was compared to that in the urinary bladder which contains almost exclusively the M2 receptors in order to determine the receptor subtypes in the uterus. $[^3H]QNB$ binding to uterus and bladder was rapid, saturable and reversible. Scatchard analysis of the saturation data gave linear plots and the Hill coefficients were close to unit, which indicated that each preparation contained a single population of specific binding sites for $[^3H]QNB$. The KD values(120 pM) for QNB were almost identical in both organs, whereas the $B_{max}$ value of 256 fmol/mg protein in the uterus was significantly different from that of 563 fmol/mg protein in the bladder. Muscarinic agonists and antagonists inhibited in a competitive manner the $[^3H]QNB$ binding to the same extent in both organs. The competition binding studies using antagonists(atropine and pirenzepine) exhibited a single binding site and this site had a low affinity for pirenzepine with the Ki value of about 330 nM. However, high and low affinity binding sites were observed with carbachol, methacholine and oxotremorine. These binding studies with agonists and antagonists did not show any differences in drug affinities between uterus and bladder. These results indicate that the muscarinic receptors in the uterus are M2 receptors which have a low affinity for pirenzepine.

• Development and Reproduction
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• v.26 no.2
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• pp.49-58
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• 2022

The differentiation and development of preadipocyte into mature adipocyte are regulated by transcription factors, such as CCAAT enhancer binding protein (Cebp) gene family and sterol regulatory element binding transcription factor 1 (Srebp1). Steroid hormones give influences on the development and function of adipocyte. The present research examined expression patterns of CCAAT enhancer binding protein alpha (Cebpa), CCAAT enhancer binding protein beta (Cebpb), CCAAT enhancer binding protein gamma (Cebpg), sterol regulatory element binding transcription factor 1 (Srebp1), androgen receptor (Ar), and estrogen receptors (Esr) among different epididymal fat parts during postnatal period by quantitative real-time polymerase chain reaction. In the distal epididymal fat, expression of Cebpa, Cebpb, Cebpg, Srebp1, Ar, and Esr2 was increased until 12 months of age, while expression of Esr1 was decreased at 5 months of age and was not detectable after 8 months of age. In the proximal epididymal fat, transcript levels of Cebps and Srebp1 were increased at 8 months of age, followed by decreases of Cebpb and Cebpg transcript levels at 12 months of age. An additional increase of Srebp1 expression was observed at 12 months of age. Expression of Ar and Esr2 were increased until 8 months of age, followed by a drop of Ar expression level at 12 months of age. Expression pattern of Esr1 was similar to that in the distal epididymal fat. In the tail epididymal fat, expression of Cebpa, Cebpg, Srebp1, Ar, and Esr2 was increased with age. Esr1 was not detectable at all. The highest level of Cebpb was observed at 8 months of age. These data suggest the possibility of developmental and functional differentiation among the epididymal fat parts.

• Molecules and Cells
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• v.37 no.3
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• pp.264-269
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• 2014

The molecular interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins plays an essential role in the transcription-independent apoptotic pathway of p53. In this study, we investigated the binding of p53 DNA-binding domain (p53DBD) with the anti-apoptotic Bcl-2 family proteins, Bcl-w, Mcl-1, and Bcl-2, using GST pull-down assay and NMR spectroscopy. The GST pull-down assays and NMR experiments demonstrated the direct binding of the p53DBD with Bcl-w, Mcl-1, and Bcl-2. Further, NMR chemical shift perturbation data showed that Bcl-w and Mcl-1 bind to the positively charged DNA-binding surface of p53DBD. Noticeably, the refined structural models of the complexes between p53DBD and Bcl-w, Mcl-1, and Bcl-2 showed that the binding mode of p53DBD is highly conserved among the anti-apoptotic Bcl-2 family proteins. Furthermore, the chemical shift perturbations on Bcl-w, Mcl-1, and Bcl-2 induced by p53DBD binding occurred not only at the p53DBD-binding acidic region but also at the BH3 peptide-binding pocket, which suggests an allosteric conformational change similar to that observed in Bcl-$X_L$. Taken altogether, our results revealed a structural basis for a conserved binding mechanism between p53DBD and the anti-apoptotic Bcl-2 family proteins, which shed light on to the molecular understanding of the transcription-independent apoptosis pathway of p53.

• YAKHAK HOEJI
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• v.46 no.6
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• pp.416-425
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• 2002

A comparative study to validate the reliability of a fully automated docking program, FlexiDock, was carried out to predict the binding modes of DHFR-inhibitor complex. The inhibitors were extracted from the crystallographically determined DHFR-NADP$^{+}$(H)-inhibitor ternary complexes of human, Escherichia coli and Candida albicans and then docked back into the remaining DHFR-NADP$^{+}$(H) binary complexes using FlexiDock. The resulting conformations and orientations were compared to the original crystal complex structures for reproducibility. Then, folate, the substrate, and known inhibitors such as methotrexate, piritrexim and trimethoprim were docked into the wild-type human DHFR and their binding modes were compared with X-ray crystallographic or other modeling data. The root mean square deviations (RMSDs) for ligands ranged from 1.14 to 1.57$\AA$, and the protein backbone RMSDs from 0.94 to 1.26$\AA$. FlexiDock reproduced the orientations and binding modes of all seven ligands in good agreement with the crystal structures. It proved to be a reliable and efficient program in studying binding modes of DHFR-inhibitor complexes of different species, and the information obtained from this work may provide additional insight into the design of new agents with improved activity.ity.

• BMB Reports
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• v.34 no.1
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• pp.1-7
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• 2001

Structural and physical properties of type wild type and various selected mutants of the vnd/NK-2 homeodomain, the protein product of the homeobox, and the implication in genetic disease are reviewed. The structure, dynamics and thermodynamics have been Investigated by NMR and by calorimetry. The interactions responsible for the nucleotide sequence-specific binding of the homeodomain to its consensus DNA binding site have been identified. There is a strong correlation between significant structural alterations within the homeodomain or its DNA complex and the appearance of genetic disease. Mutations in positions known to be important in genetic disease have been examined carefully For example, mutation of position 52 of vnd/NK-2 results in a significant structural modification and mutation of position 54 alters the DNA binding specificity and amity The $^{15}N$ relaxation behavior and heteronuclear Overhauser effect data was used to characterize and describe the protein backbone dynamics. These studies were carried out on the wild type and the double mutant proteins both in the free and in the DNA bound states. Finally, the thermodynamic properties associated with DNA binding are described for the vnd/NK-2 homeodomain. These thermodynamic measurements reinforce the hypothesis that water structure around a protein and around DNA significantly contribute to the protein-DNA binding behavior. The results, taken together, demonstrate that structure and dynamic studies of proteins combined with thermodynamic measurements provide a significantly more complete picture of the solution behavior than the individual studies.

• Journal of Nutrition and Health
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• v.32 no.4
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• pp.369-375
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• 1999

Hyperthroidism in known to alter the activity of a number of enzymes involved in the catabolism of histidine to CO2. 10-Formyltetrahydrofolate dehydrogenase(EC 1.5, 1.6, 10-formyl-THE dehydrogenase) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In previous studies, 10-formyl-THF dehydrogenase purified from rat and pig liver was coidentified with the cytosolic folate-binding protein. In this study, we investigated the effects of feeding thyroid powder (TP) and thiouracil(TU) on the folate-binding properties of 10-formyl-THE dehydrogenase, the uptake of an injected dose of [3H] folate, and the metabolism of labeled folate to pteroylopoly-${\gamma}$-glutamate in rat liver. The initial hepatic uptake(24hr) of the labeled folate dose was higher in TU-rats and slightly higher in TP-rats in controls. With longer time periods, decreased hepatic uptake of labeled folate was observed in TP-animals compared to euthroid animals, and high levels of hepatic uptake of labeled folate were maintained in TU-animals. This data shows that high levels of thyroid hormone decreased the retention of folate in rat liver. Folate polygutamate chain length was shorter in TU-rats than controls, which suggests that thyroid states do not affect the ability to synthesize pteroylpolyglutamates and that folate polyglutamate might be modulated by altered folate pool size. The ability of 10-formyl-THE dehydrogenase to bind folate in rat liver was similar in both TP-and TU-rats although dehydrogenase activity was changed by thyroid sates.

• Bulletin of the Korean Chemical Society
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• v.27 no.2
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• pp.224-230
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• 2006

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. We report here the kinetic mechanism of presteady state ATP binding and hydrolysis by the Rho-RNA complex. Presteady state chemical quenched-flow technique under multiple turnover condition was used to probe the kinetics of ATP binding and hydrolysis by the Rho-RNA complex. The quenched-flow presteady state kinetics of ATP hydrolysis studies show that three ATPs are bound to the Rho-RNA complex with a rate of $4.4\;{\times}\;10^5M^{-1}s^{-1}$, which are subsequently hydrolyzed at a rate of $88s^{-1}$ and released during the initiation process. Global fit of the presteady state ATP hydrolysis kinetic data suggests that a rapid-equilibrium binding of ATP to Rho-RNA complex occurs prior to the first turnover and the chemistry step is not reversible. The initial burst of three ATPs hydrolysis was proposed to be involved in the initialization step that accompanies proper complex formation of Rho-RNA. Based on these results a kinetic model for initiation process for Rho-RNA complex was proposed relating the mechanism of ATP binding and hydrolysis by Rho to the structural transitions of Rho-RNA complex to reach the steady state phase, which is implicated during translocation along the RNA.

• Journal of Integrative Natural Science
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• v.3 no.1
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• pp.49-53
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• 2010

Molecular docking is a critical event which mostly forms Van der waals complex in molecular recognition. Since the majority of developed drugs are small molecules, docking them into proteins has been a prime concern in drug discovery community. Since the binding pose space is too vast to cover completely, many search algorithms such as genetic algorithm, Monte Carlo, simulated annealing, distance geometry have been developed. Proper evaluation of the quality of binding is an essential problem. Scoring functions derived from force fields handle the ligand binding prediction with the use of potential energies and sometimes in combination with solvation and entropy contributions. Knowledge-based scoring functions are based on atom pair potentials derived from structural databases. Forces and potentials are collected from known protein-ligand complexes to get a score for their binding affinities (e.g. PME). Empirical scoring functions are derived from training sets of protein-ligand complexes with determined affinity data. Because non of any single scoring function performs generally better than others, some other approaches have been tried. Although numerous scoring functions have been developed to locate the correct binding poses, it still remains a major hurdle to derive an accurate scoring function for general targets. Recently, consensus scoring functions and target specific scoring functions have been studied to overcome the current limitations.

• YAKHAK HOEJI
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• v.25 no.4
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• pp.161-165
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• 1981

The binding of two cephalosporins, cephalothin and cefazoline to human serum albumin(HSA) was studied by difference spectrophotometry using a spectrophotometric probe, 2-(4'-hydroxybenzeneazo) benzoic acid. The probe is strong visible absorbing material which interacts with serum albumin to give characteristic spectrophotometric peaks and provides the basis for a convenient assay to measure free and bound amounts in the presence of serum albumin and competitive drugs. The results obtained showed that the probe and cephalosporin compete for the same binding site on human serum albumin; thus the probe can be used to gauge the displacement of cephalosporins from human serum albumin. The data were interpreted on the basis of theory of multiple equilibria. The number of binding sites of human serum albumin for 2-(4'-hydroxybenzeneazo) benzoic acid(HBAB), cephalothin and cefazoline appears to be 4. By using this technique the binding constants were found as follows: HSA-HBAB, $7.89{\times}10^{4}M^{-1}$; HSA-cephalothin, $1.09{\times}10^{3}M^{-1}$ ; HSA-cefazoline, $1.21{\times}10^{3}M^{-1}$.