Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Takeru Nose Last modified date:2020.05.28

Professor / Division for Experimental Natural Science / Faculty of Arts and Science

1. Iori Maeda, Miki Kawakami, Suguru Taniguchi, Keitaro Suyama, Takeru Nose, Stability of Phe-containing Elastin-like Peptides against Proteases in Digestive Organs, Peptide Science 2019, 161-162, 2020.03.
2. Naoki Sakamoto, Daiki Tatsubo, Keiji Sato, Keisuke Tomohara, Keitaro Suyama, Iori Maeda, Takeru Nose., Development of Liposome Like Nanostructures Composed of Short Amphiphilic Elastin-Like Peptides, Peptide Science 2019, 113-114, 2020.03.
3. Keitaro Suyama, Mika Mawatari, Daiki Tatsubo, Iori Maeda, Takeru Nose, Concentration Dependent Coacervation Property of Nonlinear Elastin-derived Peptide (FPGVG)n Analogs, Peptide Science 2019, 29-30, 2020.03.
4. Xiaohui Liu, Keitaro Suyama, Junichi Shiki, Kohei Torikai, Takeru Nose, Miki Shimohigashi, Yasuyuki Shimohigashi, Bisphenol AF
Halogen bonding effect is a major driving force for the dual ERα-agonist and ERβ-antagonist activities, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2019.115274, 28, 3, 2020.02, 17β-Estradiol (E2) is a natural steroid ligand for the structurally and physiologically independent estrogen receptors (ERs) ERα and ERβ. We recently observed that CF3-containing bisphenol AF (BPAF) works as an agonist for ERα but as an antagonist for ERβ. Similar results were also observed for the CCl3-containing bisphenol designated as HPTE. Both BPAF and HPTE are comprised of a tri-halogenated methyl group in the central alkyl moiety of their bisphenol structures, which strongly suggests that halogens contribute directly to the agonist/antagonist dual biological functions. We conducted this study to investigate the structure-activity relationships by assessing together newly synthesized CF3- and CBr3-containing bisphenol E analogs (BPE-X). We first tested bisphenols for their receptor binding ability and then for their transcriptional activities. Halogen-containing bisphenols were found to be fully active for ERα, but almost completely inactive for ERβ. When we examined these bisphenols for their inhibitory activities for E2 in ERβ, we observed that they worked as distinct antagonists. The ascending order of agonist/antagonist dual biological functions was BPE-F < BPE-Cl (HPTE) ≤ BPAF < BPE-Br, demonstrating that the electrostatic halogen bonding effect is a major driving force of the bifunctional ERα agonist and ERβ antagonist activities of BPAF..
5. Keisuke Tomohara, Naoto Hasegawa, Isao Adachi, Yoshikazu Horino, Takeru Nose, Early identification of promiscuous attributes of aldose reductase inhibitors using a DMSO-perturbation assay, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2019.126815, 30, 2, 2020.01, Aldose reductase (AR) inhibitors are used clinically to treat long-term diabetic complications. Previous studies reported a series of AR inhibitory candidates, but unfortunately the mode of inhibition was poorly described due mainly to the lack of readily available methods for evaluating the specificity. The present study examined the AR inhibitory effects of novel synthetic hydantoins and their structural relatives, some of which were obtained from chemically engineered extracts of natural plants, and discovered several novel AR inhibitors with moderate inhibitory activity. The identified inhibitors were then subjected to a two-step mechanistic characterization using a detergent-addition assay and our novel dimethyl sulfoxide (DMSO)-perturbation assay. The detergent-addition assay revealed aggregation-based inhibitors, and the subsequent DMSO-perturbation assay identified nonspecific binding inhibitors. Thus, the present study demonstrates the usefulness of the DMSO-perturbation screen for identifying nonspecific binding characteristics of AR inhibitors..
6. Xiaohui Liu, Hiroki Sakai, Mitsuhiro Nishigori, Keitaro Suyama, Tasuku Nawaji, Shin Ikeda, Makoto Nishigouchi, Hiroyuki Okada, Ayami Matsushima, Takeru Nose, Miki Shimohigashi, Yasuyuki Shimohigashi, Receptor-binding affinities of bisphenol A and its next-generation analogs for human nuclear receptors, Toxicology and Applied Pharmacology, 10.1016/j.taap.2019.114610, 377, 2019.08, An endocrine-disrupting chemical Bisphenol A (BPA) binds specifically to a nuclear receptor (NR) named ERRγ. Although the importance of receptor-binding evaluation for human NRs is often stressed, the binding characteristics of so-called next-generation (NextGen) bisphenol compounds are still poorly understood. The ultimate objective of this investigation was to evaluate BPA and its NextGen analogs for their abilities to bind to 21 human NRs, the greatest members of NRs for which tritium-labeled specific ligands were available. After establishing the detailed assay conditions for each NR, the receptor binding affinities of total 11 bisphenols were evaluated in competitive binding assays. The results clearly revealed that BPA and the NextGen bisphenols of BPAF, BPAP, BPB, BPC, BPE, and BPZ were highly potent against one or more of NRs such as CAR, ERα, ERβ, ERRγ, and GR, with IC50 values of 3.3–73 nM. These bisphenols were suggested strongly to be disruptive to these NRs. BPM and BPP also appeared to be disruptive, but less potently. BPF exhibited only weak effects and only against estrogen-related NRs. Surprisingly, most doubtful bisphenol BPS was supposed not to be disruptive. The NRs to which BPA and NextGen bisphenols did not bind were RARα, RARβ, RARγ, and VDR. PPARγ, RORα, RORβ, RORγ, RXRα, RXRβ, and RXRγ, exhibited very weak interaction with these bisphenols. The ten remaining NRs, namely, ERRγ, ERβ, ERα, CAR, GR, PXR, PR, AR, LXRβ, and LXRα, showed distinctly strong binding to some bisphenols in this order, being likely to have consequential endocrine-disruption effects..
7. Keisuke Tomohara, Isao Adachi, Yoshikazu Horino, Hitoshi Kesamaru, Hitoshi Abe, Keitaro Suyama, Takeru Nose, DMSO-Perturbing Assay for Identifying Promiscuous Enzyme Inhibitors, ACS Medicinal Chemistry Letters, 10.1021/acsmedchemlett.9b00093, 10, 6, 923-928, 2019.05, In search for enzyme inhibitors, we often encounter "promiscuous" enzyme inhibitors exhibiting nonspecific binding property toward enzyme active site. Therefore, inhibitory candidates should be mechanistically characterized as early as possible in discovery processes. However, there remains a lack of highly reliable and readily available methodology to evaluate specificity of initial hits inhibitors. The present study developed and established a novel DMSO-perturbing assay to identify promiscuous enzyme inhibitors. The assay successfully identified nonspecific binding inhibitors with a broad scope, typically by the attenuation of inhibitory activity by the influence of DMSO-addition. This attenuation would be attributed to the nonspecific binding property of inhibitors toward both productive and nonproductive (nondenatured) states of enzymes in perturbation solution. This working hypothesis was supported by spectroscopic analyses of enzyme conformations and analyses of solvent effects on perturbation. Overall, these results provided a novel concept of the DMSO-perturbing assay..
8. Hitoshi Kesamaru, Keitaro Suyama, Takeru Nose, Importance of Receptor Conformations in Docking Calculation-Based Risk Assessment for Endocrine Disruptors against Estrogen Receptor α, ACS Omega, 10.1021/acsomega.9b00050, 4, 4, 6620-6629, 2019.04, Employment of appropriate receptor conformations as templates is essential for appropriate identification of the latent receptor binding ability of chemicals using in silico docking calculations. In this study, we performed docking calculations using a number of agonist- and antagonist-bound conformations of 83 estrogen receptor (ER) α-ligand-binding domains as templates to clarify the type of receptor conformations required for reasonable identification of endocrine disruptors. Our results showed that 17β-estradiol and diethylstilbestrol (ERα agonists) bound preferentially to the agonist conformations, whereas 4-hydroxytamoxifen and raloxifene (ERα antagonists) bound selectively to the antagonist conformations. We also observed that bisphenol A analogues, which are partial agonists, bound more moderately and preferentially to the agonist conformations as compared with the antagonist conformations. Additionally, the docking calculations were able to estimate biological agonist or antagonist activity of chemicals based on the receptor conformation selectivity. Furthermore, structural analyses of the ligand-binding domains and docking calculation utilizing C-terminal-truncated receptors indicated that the C-terminal regions of these domains were capable of discriminating agonists from nonagonists. These results suggest that both agonist- and antagonist-binding conformations of receptors are necessary to predict the binding affinity and biological activity of chemicals for docking calculation-based risk assessment. Furthermore, this in silico method can be beneficial for drug discovery because it is useful for rapid searching of ligands for receptors and preventing the side effects caused by unfavorable receptor binding..
9. Keisuke Tomohara, @Isao Adachi, Hitoshi Kesamaru, Takeru Nose, Characterization of alpha-Chymotrypsin Inhibitors by DMSO-Perturbing Assay, Peptide Science 2018, 107-108, 2019.03.
10. Keitaro Suyama, Daiki Tatsubo, Suguru Taniguchi, Iori Maeda, and Takeru Nose, Development of self-assembling short elastin-derived peptide analogs: linear and nonlinear (FPGVG)n analogs, Peptide Science 2018, 117-117, 2019.03.
11. Daiki Tatsubo, Keitaro Suyama, Iori Maeda, and Takeru Nose, Structure and Function of the Elastin-like Short Peptide Analogs with Shuffled Sequences Based on (FPGVG)5, Peptide Science 2018, 65-65, 2019.03.
12. The purpose of the study is to investigate the change of awareness of student during the “KIKAN Education Seminar” for first year students in Kyushu University. The “KIKAN Education Seminar” first started under the semester system in April 2014 and had been reconstructed in response to the shift to the quarter system in April 2017. We developed new units and combined several units, while maintaining the educational purpose of the seminar. In order to evaluate the educational effects (including the change of student’s awareness), we surveyed the change of student’s attitudes from 2016 to 2017 using a questionnaire. This questionnaire focused on their learning motivation, communication skills, perspective on the future, and view of the significance of university learning. Typical results of the questionnaire and text-mining analysis concerning learning beliefs clarified that the score in 2017 about the significance of learning in university improved over 2016. The acquirement of new learning beliefs may be related to newly introduced contents. These results show the value of this seminar in the first year experience and the improvement of the quality of education due to the shift to the quarter system. We re-found the significance of this seminar via this research..
13. The researchers analyzed faculty development events (FDs) held by the Center for the Future Development of Education (hereafter called “CFDE”) in 2017, in combination with the survey of FDs held throughout the whole Kyushu University. The main purpose of this report is to reveal the role of FDs held by CFDE in comparison with those by other sections of Kyushu University. In the analysis, we focused on attributes of participants, themes, dates of the FDs, and so on. CFDE held 24% of all the FDs in the entire Kyushu University. Furthermore, there was a diversity of participants in FDs held by CFDE. Also, teaching methods such as active learning were employed in many FDs. It is also suggested that holding FDs during class term could increase participation of full-time faculty members. In the last part of this report, the researchers discussed identified subjects and perspectives..
14. Keitaro Suyama, Daiki Tatsubo, Wataru Iwasaki, Masaya Miyazaki, Yuhei Kiyota, Ichiro Takahashi, Iori Maeda, Takeru Nose, Enhancement of self-aggregation properties of linear elastin-derived short peptides by simple cyclization
strong self-aggregation properties of cyclo[FPGVG]n, consisting only of natural amino acids, Biomacromolecules, 10.1021/acs.biomac.8b00353, 19, 8, 3201-3211, 2018.06, Elastin-like peptides (ELP) consist of distinctive repetitive sequences, such as (VPGVG)n, exhibit temperature-dependent reversible self-assembly (coacervation), and have been considered to be useful for the development of thermo-responsive materials. Further fundamental studies evaluating coacervative properties of novel nonlinear ELPs could present design concepts for new thermo-responsive materials. In this study, we prepared novel ELPs, cyclic (FPGVG)n (cyclo[FPGVG]n, n = 1-5), and analyzed its self-assembly properties and structural characteristics. Cyclo[FPGVG]n (n = 3-5) demonstrated stronger coacervation capacity than the corresponding linear peptides. The coacervate of cyclo[FPGVG]5 was able to retain water-soluble dye molecules at 40°C, which implied that cyclo[FPGVG]5 could be employed as a base material of DDS (Drug Delivery System) matrices and other biomaterials. The results of molecular dynamics simulations and circular dichroism measurements suggested that a certain chain length was required for cyclo[FPGVG]n to demonstrate alterations in molecular structure that were critical to the exhibition of coacervation..
15. Daiki Tatsubo, Keitaro Suyama, Masaya Miyazaki, Iori Maeda, Takeru Nose, Stepwise Mechanism of Temperature-Dependent Coacervation of the Elastin-like Peptide Analogue Dimer, (C(WPGVG)3)2, Biochemistry, 10.1021/acs.biochem.7b01144, 57, 10, 1582-1590, 2018.02.
16. Iori Maeda, Shohei Kai, Suguru Taniguchi, Asako Inoue, Hujun Li, Hitoshi Kesamaru, Takeru Nose, Angiotensin I Converting Enzyme-inhibiting Peptides Purified from Elastase-degraded Elastin Prepared from Pig Aorta, Current Enzyme Inhibition, 10.2174/1573408013666170912113121, 14, 67-74, 2018.03.
17. Daiki Tatsubo, Misako Kodama, Keiji Sato, Keitaro Suyama, Iori Maeda, and Takeru Nose, Effects of Salts and pH on Coacervation of Short Elastin-Like Peptide (FPGVG)5, Peptide Science 2017, 62-63, 2018.03.
18. Hujun Li, Asako Inoue, Suguru Taniguchi, Tomohiko Yukutake, Keitaro Suyama, Takeru Nose, Iori Maeda, Multifunctional biological activities of water extract of housefly larvae (Musca domestica), PharmaNutrition, 10.1016/j.phanu.2017.09.001, 5, 4, 119-126, 2017.12, Many types of insects have been used as foods and protein sources. In this study, we investigated the usefulness of housefly larvae (Musca domestica) based on their amino acid composition and multifunctional biological activities. First, the utility of the amino acid composition of housefly larvae was evaluated by amino acid analysis. Notably, the housefly larvae contained sufficient amounts of all essential amino acids, and the amino acid composition was similar to that of hen eggs. Second, we prepared housefly larvae water extract (HLWE) using the decoction method and explored the biological activities of the extract for potential application of the extract as a functional food. HLWE showed significant antioxidant activity (75.4% at 5.00 mg/mL), angiotensin-I-converting enzyme (ACE) inhibitory activity (half-maximal inhibitory concentration [IC50] = 0.430 mg/mL), and dipeptidyl peptidase-IV (DPP-IV) inhibitory activity ([IC50] = 3.52 mg/mL). We found that the low-molecular-weight constituents (<6 kDa) in HLWE contributed to antioxidant and ACE-inhibitory activities, whereas the high-molecular-weight constituents (>6 kDa) contributed to DPP-IV inhibition. Our results suggested that housefly larvae may provide a useful source of multifunctional protein..
19. Keitaro Suyama, Hitoshi Kesamaru, Daiki Tatsubo, Takeru Nose, Coacervation Property and Structural Analysis of Cyclic Analogs of Elastin-derived Peptide (FPGVG)n , Peptide Science 2016, 101-102, 2017.03.
20. Daiki Tatsubo, Keitaro Suyama, Takeru Nose, Analysis of structural requirement for coacervation property of elastin model peptide dimers, Peptide Science 2016, 13-14, 2017.03.
21. Asako Inoue, Tomohiro Hikima, Suguru Taniguchi, Takeru Nose, Iori Maeda, Investigation of water-soluble elastin as a multifunctional cosmetic material: Moisturizing and whitening effects, JOURNAL OF COSMETIC SCIENCE, 10.1002/psc.2837, 68, 1-13, 2017.01.
22. 山口容子, Steve Peigneur, 劉 俊驛, Shiho Uemura, Takeru Nose, Selvanayagam Nirthanan, Ponnampalam Gopalakrishnakone, Jan Tytgat, 佐藤一紀, Role of individual disulfide bridges in the conformation and activity of spinoxin (α-KTx6.13), a potassium channel toxin from Heterometrus spinifer scorpion venom, Toxicon, 10.1016/j.toxicon.2016.09.013, 122, 31-38, 2016.11.
23. Steve Peigneur, 山口容子, Chihiro Kawano, Takeru Nose, Selvanayagam Nirthanan, Ponnampalam Gopalakrishnakone, Jan Tytgat, 佐藤一紀, Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning, Biochemistry, 10.1021/acs.biochem.6b00139, 55, 21, 2927-2935, 2016.05.
24. Keitaro Suyama, Suguru Taniguchi, Daiki Tatsubo, Iori Maeda, Takeru Nose, Dimerization effects on coacervation property of an elastin-derived synthetic peptide (FPGVG)5, Journal of Peptide Science, 10.1002/psc.2876, 22, 236-243, 2016.03.
25. Keitaro Suyama, Hitoshi Kesamaru, Daiki Tatsubo, Takeru Nose, Coacervation Properties and Structural Analysis of Aminobenzoyl-labeled Fluorescent Elastin-derived Peptides, Peptide Science 2015, 293-294, 2016.03.
26. Daiki Tatsubo, Keitaro Suyama, Takeru Nose, Fluorescence Analysis Using a Molecular Probe 1,8-ANS for Elucidation of the Molecular Mechanisms Underlying Coacervation of a Tryptophan-containing Elastin derived Dimeric Peptide, Peptide Science 2015, 95-96, 2016.03.
27. Suguru Taniguchi, Noriko Watanabe, Takeru Nose, Iori Maeda, Development of short and highly potent self-assembling elastin-derived pentapeptide repeats containing aromatic amino acid residues, JOURNAL OF PEPTIDE SCIENCE, 10.1002/psc.2837, 22, 1, 36-42, 2016.01.
28. Iori Maeda, Suguru Taniguchi, Noriko Watanabe, Asako Inoue, Yuko Yamazaki, Takeru Nose, Design of Phenylalanine-Containing Elastin-Derived Peptides Exhibiting Highly Potent Self-Assembling Capability , Protein & Peptide Letters, 10.2174/092986652210150821170703, 22, 10, 939-939, 2015.08, In this study, we developed a series of Phe-containing elastin-derived peptide-analogs, (Phe-Pro-Gly-Val-Gly)n (n = 1–5) and analyzed their reversible coacervation properties. Compared to the native elastin-derived repeating peptide sequence ((Val-Pro-Gly-Val-Gly)10), one of the Phecontaining 5-mer repeating peptide sequences ((Phe-Pro-Gly-Val-Gly)5) clearly exhibited stronger coacervation properties. The coacervation of (Phe-Pro-Gly-Val-Gly)5 is nearly the same as that of polypeptides (Val-Pro-Gly-Val-Gly)n (n > 40). Although large molecular weights (>10,000 Da) are generally required for the coacervation of elastin-derived peptides, (Phe-Pro-Gly-Val-Gly)5 exhibited reversible coacervation properties despite its low molecular weight (MW = 2,305 Da). High performance liquid chromatography (HPLC) and circular dichroism (CD) analysis revealed that (Phe-Pro-Gly-Val-Gly)5 has high hydrophobicity and an ordered structure with a type II β-turn, which contributes to the strong coacervation ability of the peptide. In addition, (Phe-Pro-Gly-Val-Gly)5 exhibited an effective particle size distribution (60–70 nm) at body temperature (37°C) and a dispersed small particle size similar to that of the monomer peptides at low temperatures. These properties, along with its small size and simple design, render the peptide suitable for use in biomaterials, including drug-delivery carriers..
29. Steve Peigneur, 山口容子, Chihiro Kawano, Takeru Nose, Selvanayagam Nirthanan, Ponnampalam Gopalakrishnakone, Jan Tytgat, 佐藤一紀, Structure-function relationships of spinoxin, a peptide neurotoxin from scorpion venom, Peptide Science 2014, 55-56, 2015.03.
30. Keitaro Suyama, Daiki Tatsubo, Suguru Taniguchi, Hitoshi Kesamaru, Iori Maeda, Takeru Nose, Coacervation property and structural analysis of synthetic dimer peptides of aromatic amino acid containing elastin-derived peptides, Peptide Science 2014, 323-324, 2015.03.
31. Iori Maeda, Suguru Taniguchi, Takeru Nose, Identification of an Elastin-derived Peptide Degrade in Human Blood After Oral Ingestion of Elastin, Peptide Science 2014, 305-306, 2015.03.
32. KIKAN education KADAI-KYOGAKU (interdisciplinary collaborative learning of social issues).
33. KIKAN education KADAI-KYOGAKU (interdisciplinary collaborative learning of social issues).
34. Keitaro Suyama, Suguru Taniguchi, Iori Maeda, Daiki Tatsubo, Takeru Nose, Coacervation Property and Secondary Structure of Synthetic Dimer Peptides of Elastin-Derived Pentapeptide Repeats, Peptide Science 2013, 277-278, 2014.03.
35. Yumi Kuramitsu, Hirokazu Nishimura, Ryo Nakamura, Keitaro Suyama, Ayami Matsushima, Takeru Nose, Yasuyuki Shimohigashi, Structure-Activity Studies on the Halogenated Phe-containing Neuropeptide Substance P Analogs, Peptide Science 2013, 319-320, 2014.03.
36. Asako Inoue, Suguru Taniguchi, Yuko Yamasaki, Masakazu Furuta, Takeru Nose, Iori Maeda, Preparing of Phenylalanine Containing Elastin-Derived Pentapeptide Based Biomaterials , Peptide Science 2013, 431-432, 2014.03.
37. Suguru Taniguchi, Noriko Watanabe, Takao Hattori, Taishi Inatomi, Asako Inoue, Yuko Yamasaki, Takeru Nose, Iori Maeda, Coacervation Properties of Short Elastin-Derived Pentapeptide Analogues Containing Aromatic Amino Acids, Peptide Science 2013, 433-434, 2014.03.
38. Iori Maeda, Suguru Taniguchi, Junko Ebina, Noriko Watanabe, Takao Hattori, Takeru Nose, Comparison between Coacervation Property and Secondary Structure of Synthetic Peptides, Ile-containing Elastin-derived Pentapeptide Repeats, Protein & Peptide Letters, 10.2174/0929866511320080007 , 20, 8, 905-910, 2013.08, A series of Ile-containing elastin-derived peptide-analogs, (Ile-Pro-Gly-Val-Gly)n (n=7–10) possessing remarkable and reversible coacervation property were newly synthesized. In comparison with the known elastin-derived peptideanalogs, which were so-called polypeptides, the obtained 35 to 50 mer peptides, (IPGVG)n (n=7–10) were significantly low molecular sized-polypeptides. However, they clearly exhibited coacervation property as same as the polypeptides did. Because of their low molecular size, spectrographic analyses of (IPGVG)n (n=7–10) became feasible to carry out. As results of secondary structural analyses by CD and FT-IR, it was found that the coacervation property of the peptides is clearly attributed to the ordered secondary-structures, mainly, type II β–turn..
39. Nishio K, Nishimura H, Suyama K, Matsushima A, Nose T, Shimohigashi, Halogenated Phe-containing endomorphin-2 analogs with mixed agonist and antagonist activities, Peptide Science 2012, 25-26, 2013.03.
40. Y. Kuramitsu, H. Nishimura, R. Nakamura, K. Suyama, A. Matsushima, T. Nose, Y. Shimohigashi, High-precision binding assay procedure of tachykinin receptor NK-1 for highly potent Substance P analogs, Peptide Science 2012, 213-214, 2013.03.
41. Maniwa Y., Inoue A., Watanabe N., Ogino Y., Yamasaki Y., Nose T., Maeda I., Coacervation properties of hydrophobic elastin-derived pentapeptide analogues, Peptide Science 2012, 391-392, 2013.03.
42. Y. Matsuyama, X. Liu, H. Nishimura, A. Matsushima, T. Nose, Y. Shimohigashi, Bisphenol-binding pocket of constitutively active nuclear receptor CAR: Docking modeling for close-packing, Peptide Science 2012, 401-402, 2013.03.
43. Mitsuhiro Nishigori, Takeru Nose, Yasuyuki Shimohigashi, Highly Potent Binding and Inverse Agonist Activity of Bisphenol A Derivatives for Retinoid-related Orphan Nuclear Receptor RORg, Toxicol. Lett.,, 212, 2, 205-211, 2012.05.
44. Asanomi Y, Koga Y, Miyazaki M, Nose T, Kodama H, and Maeda H., Protease-immobilized Microreactor for Rapid and Site-specific Affinity Tag Cleavage, Peptide Science 2011, 395-396, 2012.03.
45. Nishimura H, Li J, Isozaki K, Abe Y, Inamine S, Matsushima A, Nose T, Costa T, and Shimohigashi Y, Structural Essentials of Hyperalgesic Nociceptin ORL1 Receptor for Ligand Binding and Receptor Activation, Peptide Science 2011, 33-34, 2012.03.
46. Nakamura R, Nishimura H, Suyama K, Nose T, and Shimohigashi Y, The Effects of Halogenation of Phe-phenyl Group of Two Consecutive Phe Residues Present in Neuropeptide Substance P on its Specific Receptor Interaction, Peptide Science 2011, 157-158, 2012.03.
47. Nishio K, Nishimura H, Suyama K, Abe Y, Matsushima A, Nose T, Shimohigashi Y, Effects of the Halogenation of Phe-phenyl Group of Two Consecutive Residues in endomorphin-2 on the Interaction with the μ-opioid receptors, Peptide Science 2011, 171-172, 2012.03.
48. Ito N, Nishimura H, Matsushima A, Nose T, Costa T, and Shimohigashi Y, Structural Analysis of Delta Opioid Receptor Dimer by Bivalent Deltorphin II Analogs, Peptide Science 2011, 173-174, 2012.03.
49. Inamine S, Nishimura H, Li J, Matsushima A, Nose T, Costa T, and Shimohigashi Y, Receptor-binding Characteristics of Tritium-labeled Pure Antagonist Peptide for Hyperalgesic Nociceptin ORL1 Receptor

 , Peptide Science 2011, 183-184, 2012.03.
50. Abe Y, Matsuo T, Nishimura H, Li J, Nose T, Costa T, and Shimohigashi Y, Functional Analysis of a Histidine Residue Essential for Receptor Activation of Delta Opioid Receptor

 , Peptide Science 2011, 185-186, 2012.03.
51. Matsuo A, Nishimura H, Nakamura M, Koga K, Sumiyoshi M, Matsushima A, Nose T, Shimohigashi M, and Shimohigashi Y, Structural Characteristics of Thr-Gly Dipeptide Repeat in the Drosophila Clock Protein PERIOD, Peptide Science 2011, 351-352, 2012.03.
52. Liu X., Matsushima A., Nakamura M., Costa T., Nose T., and Shimohigashi Y., Fine Spatial Assembly for Construction of the Phenol Binding Pocket to Capture Bisphenol A in the Human Nuclear Receptor ERRγ, J. Biochem.  , 10.1093/jb/mvs008, 151, 4, 403-415, 2012.01, [URL], Various lines of evidence have shown that bisphenol A (BPA) acts as an endocrine disruptor that affects various hormones even at merely physiological levels. We demonstrated recently that BPA binds strongly to human nuclear receptor estrogen-related receptor γ (ERRγ), one of 48 nuclear receptors. Based on X-ray crystal analysis of the ERRγ ligand-binding domain (LBD)/BPA complex, we demonstrated that ERRγ receptor residues, Glu275 and Arg316, function as the intrinsic-binding site of the phenol–hydroxyl group of BPA. If these phenol–hydroxyl↔Glu275 and Arg316 hydrogen bonds anchor the A-benzene ring of BPA, the benzene–phenyl group of BPA would be in a pocket constructed by specific amino acid side chain structures. In the present study, by evaluating the Ala-replaced mutant receptors, we identified such a ligand-binding pocket. Leu268, Leu271, Leu309 and Tyr326, in addition to the previously reported participants Glu275 and Arg316, were found to make a receptacle pocket for the A-ring, whereas Ile279, Ile310 and Val313 were found to assist or structurally support these residues. The results revealed that each amino acid residue is an essential structural element for the strong binding of BPA to ERRγ..
53. Maeda I., Fukumoto Y., Nose T., Shimohigashi Y., Nezu T., Terada Y., Kodama H., Kaibara K., and Okamoto K., Structural Requirements Essential for Elastin Coacervation: Favorable Spatial Arrangements of Valine Ridges on the Three-dimensional Structure of Elastin-derived Polypeptide (VPGVG)n, J. Pept. Sci., 10.1002/psc.1394, 17, 11, 735-743, 2011.06, [URL], The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3–9 mer sequences. One of these is the pentapeptide Val-Pro-Gly-Val-Gly (VPGVG) present in almost all animal species, and its polymer (VPGVG)n coacervates just as does tropoelastin. In the present study, in order to explore the structural requirements essential for coacervation, (VPGVG)n and its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated. In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings. The Gly5-deleted polymer (VPGV)n also achieved self-association, though the onset of self-association occurred at a lower temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three-step process; the Vali4-Vali+11 structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self-association was observed for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT-IR, and 1H-NMR showed that the (VPGV)n and (VPG)n both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element essential to achieving the β-spiral structure required for self-association followed by coacervation, probably due to the ideal spatial arrangement of the hydrophobic Val residues. .
54. Nishimura H., Li J., Inokuchi N., Koikawa S., Nose T., Matsushima A., Costa T., and Shimohigashi Y., A Trp Residue of Opioid Receptor TM5 is Present at the Cell Membrane Interface as a Molecular Anchor for Full Activation, Peptide Science 2010, 175, 2011.03.
55. Inamine S, Li J, Nishimura H, Matsushima A, Nose T, Costa T, and Shimohigashi Y, Exploration of the Binding Site of ORL1 Nociceptin Receptor Antagonist, Peptide Science 2010, 167, 2011.03.
56. Nishimura H., Li J., Inokuchi N., Koikawa S., Matsushima A., Nose T., Costa T., Shimohigashi Y., The functional role of Trp present at the cell membrane interface in the opioid receptor activation, Peptide Science 2009, 23-24 (2010), 2010.03.
57. Li J., Isozaki K., Matsushima A., Nose T., Costa T., Shimohigashi Y., Structure-function analysis of nociceptin receptor ORL1 by the site-directed mutagenesis, Peptide Science 2009, 219-222 (2010), 2010.03.
58. Nose T., Tokunaga T., Shimohigashi Y., The agonist/antagonist differential-docking screening (AADS) method for exploration of the estrogen receptor-binding chemicals, Peptide Science 2009, 463-464 (2010), 2010.03.
59. Isozaki K., Li J., Okada K., Nishimura H., Matsushima A., Nose T., Costa T., Shimohigashi Y., Spare Interactions of Highly Potent [Arg14,Lys15]nociceptin for Cooperative Induction of ORL1 Receptor Activation, Bioorg. Med. Chem.,, 17, 23, 7904-7908, 2009.12.
60. Nose T., Tokunaga T., and Shimohigashi Y., Exploration of endocrine-disrupting chemicals on estrogen receptor α by the agonist/antagonist differential-docking screening (AADS) method: 4-(1-Adamantyl)phenol as a potent endocrine disruptor candidate, Toxicol. Lett.,, in press, 2009.11.
61. Nishimura H., Li J., Isozaki K., Okada K., Matsushima A., Nose T., Costa T., Shimohigashi Y., Discriminatory synergistic effect of Trp-substitutions in superagonist [(Arg/Lys)(14), (Arg/Lys)(15)]nociceptin on ORL1 receptor binding and activation., Bioorg. Med. Chem.,, 17(15), 5683-5687 (2009)., 2009.08.
62. Li J., Isozaki K., Nose T., Costa T., Shimohigashi Y., Essential Amino Acid Residues in the ORL1 Receptor Transmembrane Domains for Receptor Activation, Peptide Science 2008, 33-34 (2009), 2009.01.
63. Matsuo T., Abe Y., Li J., Isozaki K., Nose T., Shimohigashi Y., Histidines as Structurally Essential Residue for the Delta Opioid Receptor Activation, Peptide Science 2008, 263-264 (2009), 2009.01.
64. Koga K., Nose T., Horiuchi Y., Shimohigashi Y., Molecular Mechanism of alpha-Helix Peptide That Inhibits Intermolecular Interaction of Prion Protein N-Terminal Tetrarepeaat Domain, Peptide Science 2008, 265-266 (2009), 2009.01.
65. Nishimura H., Li J., Isozaki K., Okada K., Nose T., Costa T., Shimohigashi Y., The Effects of Arg→Trp and Lys →Trp Substitutions for Arg-Lys14-15 Residues in a Superagonist [Arg-Lys14-15]-Nociceptin on the ORL1 Receptor Binding and Activation, Peptide Science 2008, 267-268 (2009), 2009.01.
66. Yokotani S., Nose T., Horiuchi Y., Matsushima A., and Shimohigashi Y., Radar Chart Deviation Analysis of Prion Protein Amino Acid Composition Defines Characteristics Structural Abnomalities of the N-Terminal Octapeptide Tandem Repeat, Protein Pept. Lett.,, 15(9), 949-955 (2008)., 2008.10.
67. Li J., Isozaki K., Okada K., Matsushima A., Nose T., Costa T., and Shimohigashi Y., Designed Modification of Partial Agonist of ORL1 Nociceptin Receptor for Conversion into Highly Potent Antagonist, Bioorg. Med. Chem.,, 16(5), 2635-2644 (2008)., 2008.10.
68. Okada K., Isozaki K., Li J., Matsushima A., Nose T., Costa T., and Shimohigashi Y., Synergistic Effect of Basic Residues at Positions 14-15 of Nociceptin on Binding Affinity and Receptor Activation, Bioorg. Med. Chem.,, 16(20), 9261-9267 (2008)., 2008.09.
69. Takeru Nose, Yasuyuki Shimohigashi, A docking modelling rationally predicts strong binding of bisphenol A to estrogen-related receptor γ , Protein and Peptide Letters, 15, 290-296, 2008.03.
70. M. Nishigori, T. Nose, X. Liu, T. Tokunaga, Y. Shimohigashi, The Conformation Change-Sensing Antibodies for Retinoid-Related Orphan Receptor Family, Peptide Science 2007, 491-492, 2008.03.
71. N. Inokuchi, K. Isozaki, Y. Tsuda, Y. Okada, S. Osada, T. Nose, T. Costa, Y. Shimohigashi, Differential Receptor Recognition by Dmt-Containing Enlephalin Dimers Cross-Linked by Phenylenediamines, Peptide Science 2007, 303-306, 2008.03.
72. K. Isozaki, J. Li, T. Nose, T. Costa, Y. Shimohigashi, The Molecular Mechanism of ORL1 Nociceptin Receptor in Activation: Residual Essential in the Sixth Transmembrane Domain, Peptide Science 2007, 289-292, 2008.03.
73. Y. Horiuchi, T. Nose, Y. Abe, Y. Shimohigashi, F. Morishita, Structural Analysis of Excitatory Neuropeptides TEP-1 and TEP-2 Isolated from the Prosobraanch Gastopod Thais clavigera, Peptide Science 2007, 273-276, 2008.03.
74. J. Li, K. Isozaki, A. Matsushima, T. Nose, T. Costa, Y. Shimohigashi, Optimization of the N-terminal Group of Ac-RYYRIL-NH2 as ORL1 Receptor Antagonist, Peptide Science 2007, 257-260, 2008.03.
75. I. Maeda, Y. Fukumoto, T. Nose, Y. Shimohigashi, T. Nezu, Y. Terada, H. Kodama, K. Kaibara, K. Okamoto, Structural Profiles for Coaservation of Elastin-derived Polypeptides, Peptide Science 2007, 219-222, 2008.03.
76. Jinglan Li, Kaname Isozaki, Kazushi Okada, Ayami Matsushima, Takeru Nose, Tommaso Costa and Yasuyuki Shimohigashia, Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist, Bioorganic and Medicinal Chemistry, 16, 2635-2644, 2007.11.
77. Takeshi Honda, Naoto Shirasu, Kaname Isozaki, Michiaki Kawano, Daiki Shigehiro, Yoshiro Chuman, Tsugumi Fujita, Takeru Nose, Yasuyuki Shimohigashi, Differential receptor binding characteristics of consecutive phenylalanines in μ-opioid specific peptide ligand endomorphin-2, Bioorganic and Medicinal Chemistry, 10.1016/j.bmc.2007.03.009, 15, 11, 3883-3888, 2007.06, Endogenous opioid peptides consist of a conserved amino acid residue of Phe3 and Phe4, although their binding modes for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the μ opioid receptor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe3 and Phe4 in binding to the μ receptor, we synthesized a series of analogs in which Phe3 and Phe4 were replaced by various amino acids. It was found that the aromaticity of the Phe-β-phenyl groups of Phe3 and Phe4 is a principal determinant of how strongly it binds to the receptor, although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe3 and Phe4 of endomorphin-2 were found to exhibit different structural requirements. The results suggest that [Trp3]endomorphin-2 (native endomorphin-1) and endomorphin-2 bind to different receptor subclasses..
78. K. Isozaki, K. Okada, S. Koikawa, T. Nose, T. Costa, and Y. Shimohigashi, Residual Roles of Hydrophobic Amino Acids in the Fifth Transmembrane Domain of ORL1 Receptor in Its Activation, Peptide Science 2006, 11, 2006.11.
79. J. Li, K. Isozaki, K. Okada, T. Nose, T. Costa, and Y. Shimohigashi, Synthesis of Pure Antagonist of ORL1 Nociceptin Receptor, Peptide Science 2006, 175, 2006.11.
80. T. Tokunaga, X. Liu, H. Okada, A. Mastushima, T. Nose, M. Shimohigashi, and Y. Shimohigashi, Conformation Change of α-Helix Peptide for Sensing of Deactivation of Nuclear Receptor: Immunoassay Using Polyclonal Antibody Specific for the C-terminal α-Helix 12 of Estrogen-related Receptor γ (ERRγ) , Peptide Science 2006, 177, 2006.11.
81. T. Tokunaga, H. Okada, T. Nose, and Y. Shimohigashi, Conformation Sensing Assay Using Polyclonal Antibody Specific for the C-terminal α-helix of Glucocorticoid Receptor and Progesterone Receptor, Peptide Science 2005, 291-294, 2006.03.
82. H. Okada, T. Tokunaga, N. Shirasu, T. Nose, and Y. Shimohigashi, α-Helix peptides for bio-panning in the phage display method to obtain the antibodies specific for conformation-change in nuclear receptors, Peptide Science 2005, 475-478, 2006.03.
83. K. Isozaki, J. Funama, T. Nose, T. Coosta, and Y. Shimohigashi, Continuous Activation of the δ-opioid Receptor by Affinity Labeling, Peptide Science 2005, 475-478, 2006.03.
84. J. Funama, K. Isozaki, T. Nose, T. Coosta, and Y. Shimohigashi, Enkephalin Dimers Cross-linked by Diaminobenzene for Homodimeric Opioid Receptors Expressed in COS-7 Cells, Peptide Science 2005, 475-478, 2006.03.
85. A. Matsushima, S. Yokotani, X.H. Lui, K. Sumida, T. Honda, S. Sato, A. Kaneki, Y. Takeda, Y. Chuman, M. Ozaki, D. Asai, T. Nose, H. Onoue, Y. Ito, Y. Tominaga, Y. Shimohigashi, and M. Shimohigashi, Molecular cloning and circadian expression profile of insect neuropeptide PDF in black blowfly, Phormia regina, Lett. Peptide Sci., 10.1007/BF02442573, 10, 5-6, 419-430, 10 (5-6), 419-430, 2004.05.
86. A. Matsushima, S. Sato, Y. Chuman, Y. Takeda, S. Yokotani, T. Nose, Y. Tomioka, M. Shimohigashi, and Y. Shimohigashi, cDNA Cloning of the Housefly Pigment-Dispersing Factor (PDF) Precursor Protein and Its Peptide Comparison among the Insect Circadian Neuropeptides, J. Peptide Sci., 10.1002/psc.511, 10, 2, 82-91, 10 (2) 82-91, 2004.02.
87. Y. In, S. Kishima, K. Minoura, T. Nose, Y. Shimohigashi, and T. Ishida, Aggregation feature of fluorine-substituted benzene rings and intermolecular C-H center dot center dot center dot F interaction: Crystal structure analyses of mono- and trifluoro-L-phenylalanines, Chem. Pharm. Bull.,, 10.1248/cpb.51.1258, 51, 11, 1258-1263, 51 (11), 1258-1263, 2003.11.
88. K. Isozaki, H. Fukahori, T. Honda, N. Shirasu, K. Okada, T. Nose, K. Sakaguchi, and Y. Shimohigashi, Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues, Lett. Peptide Sci.,, 10.1007/BF02442583, 10, 5-6, 511-522, 10 (5-6), 511-522, 2003.05.
89. M. Kawano, K. Okada, T. Honda, T. Nose, K. Sakaguchi, T. Costa, and Y. Shimohigashi, Structural Requirements of Nociceptin Antagonist Ac-RYYRIK-NH2 for Receptor Binding, J. Peptide Sci.,, 10.1002/psc.415, 8, 10, 561-569, 8 (10), 561-569, 2002.10.
90. A. Tani, T. Ogawa, T. Nose, N. N. Nikandrov, M. Deshimaru, T. Chijiwa, C.-C. Chang, Y. Fukumaki, and M. Ohno, Characterization, Primary Structure and Molecular Evolution of Anticoagulant Protein from Agkistrodon actus Venom, Toxicon, 10.1016/S0041-0101(01)00289-6, 40, 6, 803-813, 40 (6) 803-813, 2002.06.
91. A. Matsushima, T. Fujita, K. Okada, N. Shirasu, T. Nose, and Y. Shimohigashi, Exploration of the Role of Phenylalanine in the Thrombin Receptor Tethered-Ligand by Substitution with a Series of Trifluorophenylalanines, Bull. Chem. Soc. Jpn., 10.1246/bcsj.73.2531, 73, 11, 2531-2538, 73(11), 2531-2538, 2000.11.
92. K. Okada, T. Sujaku, Y. Chuman, R. Nakashima, T. Nose, T. Costa, Y. Yamada, M. Yokoyama, A. Nagahisa, and Y. Shimohigashi, Highly potent Nociceptin Analog Containing the Arg-Lys Triple Repeat, Biochem. Biophys. Res. Commun., 10.1006/bbrc.2000.3822, 278, 2, 493-498, 278(2), 493-498, 2000.11.
93. H. Kawasaki, T. Nose, T. Muta, S. Iwanaga, Y. Shimohigashi, and S. Kawabata, Head-to-Tail Polymerization of Coagulin, a Clottable Protein of the Horseshoe Crab, J. Biol. Chem., 10.1074/jbc.M006856200, 275, 45, 35297-35301, 275(45), 35297-3530, 2000.11.
94. T. Honda, N. Shirasu,Y. Chuman, K. Okada, T. Fujita, T. Nose, and Y. Shimohigashi, The Role of Deltorphin II Phenylalanine Residue in Binding to the δ Opioid Receptor, Bull. Chem. Soc. Jpn., 10.1246/bcsj.73.2549, 73, 11, 2549-2552, 73(11), 2549-2552, 2000.11.
95. T. Fujita, T. Nose, A. Matsushima, K. Okada, D. Asai, Y. Yamauchi, N. Shirasu, T. Honda, D. Shigehiro, and Y. Shimohigashi, Synthesis of a Complete Set of L-difluorophenylalanines, L-(F-2)Phe, as Molecular Explorers for the CH/π Interaction between Peptide Ligand and Receptor , Tetrahedron Lett., 10.1016/S0040-4039(99)02191-7, 41, 6, 923-927, 41(6), 923-927, 2000.06.
96. A. Matsushima, T. Fujita, T. Nose, and Y. Shimohigashi, Edge-to-face CH/π Interaction between Ligand Phe-phenyl and Receptor Aromatic Group in the Thrombin Receptor Activation, J. Biochem., 128, 2, 225-232, 128(2), 225-232, 2000.02.
97. D. Asai, Y. Tahara, M. Nakai, Y. Yakabe, M. Takatsuki, T. Nose, T. Shinmyozu, and Y. Shimohigashi, Structural Essentials of Xenoestrogen Dialkyl Phthalates to Bind to the Estrogen Receptors, Toxicology Lett., 10.1016/S0378-4274(00)00253-8, 118, 1-2, 1-8, 118(1-2), 1-8 (2000)., 2000.01.
98. K. Okada, T. Sujaku, R. Nakashima, T. Nose, Y. Yamada, M. Yokoyama, A. Nagahisa, and Y. Shimohigashi, Effects of Substitution of Hydrophobic Amino Acid by Tryptophan on Receptor Binding and Biological Activity of Neuropeptide Nociceptin, Bull. Chem. Soc. Jpn., 10.1246/bcsj.72.1899, 72, 8, 1899-1904, 72(8), 1899-1904, 1999.08.
99. Y. Shimohigashi, T. Nose, Y. Yamauchi, and I. Maeda, Design of Serine Protease Inhibitors with Conformation Restricted by Amino Acid Side Chain-Side Chain CH/π Interaction, Biopolymers, 10.1002/(SICI)1097-0282(1999)51:1<9::AID-BIP3>3.0.CO;2-5, 51, 1, 9-17, 51, 9-17., 1999.06.
100. S. Tanabe, Y. Shimohigashi, Y. Nakayama, T. Makino, T. Fujita, T. Nose, G. Tsujimoto, T. Yokokura, M. Naito, T. Tsuruo, and T. Terasaki, In Vivo and In Vitro Evidences of Blood-Brain Barrier Transport of a Novel Cationic Arginine-Vasopressin Fragment 4-9 Analogue, J. Pharmacol. Exp. Ther.,, 290, 2, 561-568, 290(2), 561-568, 1999.08.
101. T. Fujita, M. Nakajima, Y. Inoue, T. Nose, and Y. Shimohigashi, A novel Molecular Design of Thrombin Receptor Antagonist, Bioorg. Med. Chem. Lett., 10.1016/S0960-894X(99)00202-4, 9, 10, 1351-1356, 9, 1351-1356, 1999.05.
102. T. Fujita, T. Nose, M. Nakajima, Y. Inoue, T. Costa, and Y. Shimohigashi, Design and Syntheses of para-Fluorophenylalanine Amide Derivatives as Thrombin Receptor Antagonist, J. Biochem., 126, 1, 174-179, 126(1), 174-179, 1999.01.
103. N. Shirasu, T. Kuromizu, H. Nakao, Y. Chuman, T. Nose, T. Costa, and Y. Shimohigashi, Exploration of Universal Cysteines in the Binding Site of Three Opioid Receptor Subtypes by Disulfide-Bonding Affinity Labeling with Chemically Activated Thiol-containing Dynorphin A Analogs, J. Biochem.,, 126, 1, 254-259, 126(1), 254-259, 1999.01.
104. Nose, T., Satoh, Y., Fujita, T., Ohno, M., Nakajima, M., Inoue, Y., Ogino, Y., Costa, T., and Shimohigashi, Y, The Role of Arginine in Thrombin Receptor Tethered-Ligand Peptide in Intramolecular Receptor Binding and Self-activation, Bull. Chem. Soc. Jpn.,, 10.1246/bcsj.71.1661, 71, 7, 1661-1665, 71(7), 1661-1665, 1998.07.
105. A. Kashima, Y. Inoue, S. Sugio, I. Maeda, T. Nose, and Y. Shimohigashi, X-ray Crystal Structure of a Dipeptide-Chymotrypsin Complex in an Inhibitory Interaction, Eur. J. Biochem., 10.1046/j.1432-1327.1998.2550012.x, 255, 1, 12-23, 255(1), 12-23, 1998.07.
106. T. Nose, T. Fujita, M. Nakajima, Y. Inoue, T. Costa, and Y. Shimohigashi, Interaction Mode of the Phe-phenyl Group of Thrombin Receptor Tethered-ligand SFLLRNP in Receptor Activation, J. Biochem., 124, 2, 354-358, 124(2), 354-358, 1998.02.
107. Yasuyuki Shimohigashi, Ryo Hatano, Tsugumi Fujita, Rie Nakashima, Takeru Nose, Tetsujo Sujaku, Aki Saigo, Katsuhiro Shinjo, Atsushi Nagahisa, Sensitivity of opioid receptor-like receptor ORL1 for chemical modification on nociceptin, a naturally occurring nociceptive peptide, Journal of Biological Chemistry, 10.1074/jbc.271.39.23642, 271, 39, 23642-23645, 1996.10, Nociceptin or orphanin FQ is a novel neuropeptide that activates an opioid-like G protein-coupled receptor ORL1. This heptadecapeptide FGGFTGARKSARKLANQ resembles κ-opioid peptide dynorphin A but exhibits an opposite effect to make animals hyperreactive to nociceptive stimulations (Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazarguil, H., Vassart, G., Parmentier, M., and Costentin, J. (1995) Nature 377, 532-535; Reinscheid, R. K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H., Monsma, F. J., Jr., and Civelli, O. (1995) Science 270, 792-794). In the present study, it was found that guinea pig brain contains receptors to which nociceptin binds much more strongly than to ORL1 receptors expressed in human 293 cells. Although the Tyr1 → Phe substitution for dynorphin A eliminates almost completely an ability to bind to opioid receptors, the Phe1 → Tyr substitution in nociceptin was found to retain almost fully both receptor binding affinity and in vivo hyperalgesic activity in tail-flick assay. Nociceptin was extremely weak to bind to opioid receptors, while Tyr1- nociceptin exhibited 10-40 times increased affinity, especially for μ receptors, due to its N-terminal sequential identity to opioid peptides. Shortened analogs of dynorphin A are known to retain receptor binding ability and analgesic activity, whereas the removal of C-terminal hexa- or decapeptide from nociceptin totally abolished the affinity for the ORL1 receptor. These results indicated that the mode of interaction between nociceptin and ORL1 receptor is quite different from that between dynorphin and opioid receptor and that the C-terminal portion of nociceptin is crucial for receptor recognition..
108. Iori Maeda, Yasuyuki Shimohigashi, Koichi Ikesue, Takeru Nose, Yuzuru Ide, Keiichi Kawano, Motonori Ohno, Chymotrypsin inhibition induced by side chain-side chain intramolecular CH/π interaction in D-Thr-L-Phe benzylamide, Journal of Biochemistry, 119, 5, 870-877, 1996.05, The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (K(i) = 4.5 x 10-6 M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH2)(n)-C6H5 (n = 0-4) were tested, inhibitory potency peaked at n = 1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH3)-C6H5, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (about 5-fold, K(i) = 9.1 x 10-7 M). In conformational analysis by 400 mHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upheld shifts of D-Thr-βOH (shifts in ppm, 0.09-0.17), D-Thr-βCH (0.23-0.32), and D-Thr-γCH3 (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-γCH3 and Phe-phenyl groups were evidenced by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/π interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1 site, while the hydrophobic core with CH/π interaction binds at the S2 or S1' site..
109. Yasuyuki Shimohigashi, Iori Maeda, Takeru Nose, Koichi Ikesue, Hiroshi Sakamoto, Tomohisa Ogawa, Yuzuru Ide, Megumi Kawahara, Takashi Nezu, Yoshihiro Terada, Keiichi Kawano, Motonori Ohno, Chymotrypsin inhibitory conformation induced by amino acid side chain-side chain intramolecular CH/π interaction, Journal of the Chemical Society - Perkin Transactions 1, 10.1039/P19960002479, 20, 2479-2485, 1996.01, Dipeptide amides H-D-Leu-Phe-NH-R have been found to assume a conformation induced by the CH/π interaction and to inhibit chymotrypsin strongly. A series of benzyl amide derivatives H-D-Leu-Phe-NH-[CH2]n-C6H5 (n = 0-4) have been assayed for chymotrypsin. They inhibit the enzyme in a competitive manner and the highest inhibition is achieved by the amide of n = 1 (Ki = 3.6 × 10-6 M). The activity enhancement is dependent upon the length of methylene chain, not upon the increase in molecular hydrophobicity, indicating the presence of an optimal distance between dipeptide backbone and C-terminal phenyl group for chymotrypsin inhibition. The C-terminal phenyl group has been found to interact with chymotrypsin stereospecifically. The R-isomer of H-D-Leu-Phe-NH-CH(CH3)-C6H5 is as active as the benzyl amide, while the S-isomer is about twenty-fold less active. When the fluorine atom is introduced at a para-position of the C-terminal phenyl group, the resulting dipeptide H-D-Leu-Phe-NH-CH2-C6H4F-p exhibits about six-times increased inhibitory activity (Ki = 6.1 × 107 M; this dipeptide is one of the most potent chymotrypsin inhibitors to date). 1H NMR conformational analyses of these dipeptide amide derivatives show the CH/π interaction between D-Leu-isobutyl and Phe-phenyl as a key structural element for chymotrypsin inhibition. These structural examinations strongly suggest that in the inhibitory conformation the C-terminal phenyl group fits the chymotrypsin S1 site, while the hydrophobic core constructed by D-Leu-Phe CH/π interaction fits the chymotrypsin S2 or S1′ site..
110. Teruo Yasunaga, Shihoko Motoyama, Takeru Nose, Hiroaki Kodama, Michio Kondo, Yasuyuki Shimohigashi, Reversible affinity labeling of opioid receptors via disulfide bonding
Discriminative labeling of μ and δ subtypes by chemically activated thiol-containing enkephalin analogs, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a021433, 120, 2, 459-465, 1996.01, The 3-nitro-2-pyridinesulfenyl (Npys) group bound to a mercapto group is a highly activated electrophilic reagent, which only reacts with a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. We incorporated the Npys group into enkephalin analogs to affinity label μ and δ opioid receptors. When rat brain membranes were incubated with [D-Ala2,Leu(CH2SNpys)5] enkephalin, and assayed for the inhibition of binding of DAGO and DSLET enkephalin analogs to opioid receptors, the number of receptors decreased sharply, depending upon the concentration of this SNpys-containing enkephalin. It was found that this enkephalin analog occupies μ receptors highly specifically (EC50, = 51 nM) and almost 100 times more selectively than δ receptors. In contrast, [D-Ala2,Leu5] enkephalyl-Cys(Npys)6 attached covalently to δ receptors (EC50 = 34 nM) about 150 times more selectively than to μ receptors. Although N-ethylmaleimide also inhibited the binding of DAGO and DSLET, four to six orders of magnitude higher concentrations were required as compared to SNpys-containing enkephalins. When enkephalin-bound rat membranes were treated with dithiothreitol, the loss of receptors was reversed, depending upon the concentration of and incubation time with dithiothreitol. The recovery was much faster (about 1,000 times) for δ receptors than for μ. receptors. The present results indicated that both μ and δ receptors in rat brain consist of a free mercapto group near the enkephalin binding site and that SNpys-containing enkephalins can label these mercapto groups discriminatively. The disulfide bond between [D-Ala2,Leu5]enkephalyl-Cys6 and δ receptors appears to be exposed, while that between [D-Ala2,Leu(CH2SNpys)5] enkephalin and μ receptors is shielded..
111. T. Nose, Y. Shimohigashi, M. Okazaki, Y. Satoh, T. Costa, N. Shimizu, Y. Ogino, M. Ohno, Different roles of two consecutive leucine residues in a receptor-tethered ligand peptide (SFLLRNP) in thrombin receptor activation, Bulletin of the Chemical Society of Japan, 10.1246/bcsj.68.2695, 68, 9, 2695-2698, 1995.09, A synthetic peptide, H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-NH2, which corresponds to a ligand peptide tethered to a human thrombin receptor, was able to activate the thrombin receptor with no thrombin. In order to inspect the structural requisites of two consecutive leucines (Leu-3 and Leu-4) in receptor activation, two sets of analogs with substitutions at either position 3 or 4 were synthesized and evaluated for their ability to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells. The replacement of Leu-4 by Ala drastically decreased the activity of the parent peptide (only about 4% activity), while that of Leu-3 retained about 50% activity. A similar result was obtained when replaced by Gly. Substitution by Phe for Leu-3 sustained full activity. Although the Leu-4/Phe substitution exhibited a slight reduction in activity, Leu-4/Ile substitution unexpectedly diminished the activity (20%). These results suggested that two consecutive leucines have different roles in receptor activation; i.e., Leu-3 behaves something like a connection of peptide units to construct a bioactive conformation and Leu-4 acts like a structural element essential for interactions with receptors..
112. Y. Shimohigashi, Takeru Nose, M. Ohno, Y. Ogino, T. Costa, Human thrombin receptors are insensitive to thrombin-like snake venom enzymes, Biochemistry and Molecular Biology International, 35, 2, 415-421, 1995.02, Thrombin-like snake venoms enzymes, flavoxobin, and okinaxobin I isolated from Trimeresurus flavoviridis and Trimeresurus okinavensis, respectively, were examined in SH-EP cells and evaluated whether or not they can activate human thrombin receptors. Flavoxobin was almost completely inactive in both assays for phosphoinositide turnover and DNA synthesis. In contrast, okinaxobin I stimulated phosphoinositide turnover in a dose dependent manner, but considerably weakly. The EC50 value was about 100 nM, which was 4,000 times larger than that of α-thrombin. This stimulation was not inhibited by hirudin, an effective inhibitor of α-thrombin. Okinaxobin I also induced a very weak stimulation of DNA synthesis. These results suggest that thrombin-like snake venom enzymes interact with human thrombin receptors in inefficient ways. Weak interactions of the enzymes with thrombin receptor and inhibitor were ascribed to the incomplete formation of a lysine-cation cluster necessary for electrostatic molecular recognition..
113. Yasuyuki Shimohigashi, Takeru Nose, Mika Okazaki, Yusuke Satoh, Motonori Ohno, Tommaso Costa, Naokata Shimizu, Yoshio Ogino, Differential roles of two consecutive phenylalanine residues in thrombin receptor-tethered ligand peptides (SFFLRNP) in thrombin receptor activation, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1994.2191, 203, 1, 366-372, 1994.08, A synthetic heptapeptide H-Ser-Phe-Phe-Leu-Arg-Asn-Pro-NH2, which corresponds to the ligand peptide latent in rodent thrombin receptors, was able to activate the thrombin receptor with no thrombin. In order to evaluate the structural requisites of two consecutive phenylalanines, three sets of analogs with substitutions at either position 2 or 3 were synthesized and examined for their stimulatory activity in phosphoinositide turnover in SH-EP epithelial-like cells. The replacement of Phe-2 by Ala completely eliminated the activity, while that of Phe-3 retained about 50% activity with a full stimulation. The Phe/Leu substitution resulted in a large increase (37-fold) in EC50 value for Phe-2, but in insignificant change for Phe-3. Substitution of para-fluorophenylalanine ((p-F)Phe) for Phe-2 enhanced strongly (4-fold) the activity, in contrast to a reduction by the Phe-3/(p-F)Phe substitution. Elimination of either Phe-2 or Phe-3 resulted in a complete loss of activity. These results indicated that Phe-2 and Phe-3 play different roles in the receptor activation. A highly specific aromatic π-π interaction was suggested between Phe-2 phenyl and thrombin receptor binding site, while Phe-3 appeared to be important for retaining a bioactive conformation..
114. K. Sakaguchi, H. Kodama, Y. Ogino, T. Costa, Takeru Nose, Y. Shimohigashi, Structural essentials of Ser-1 in tethered peptide ligand of human thrombin receptor for phosphoinositide hydrolysis, Bulletin of the Chemical Society of Japan, 10.1246/bcsj.67.1659, 67, 6, 1659-1663, 1994.08, In order to inspect the structural elements of Ser-1 in receptor activation by SFLLRNP (one-letter amino acid code), a ligand peptide tethered to the thrombin receptor, a series of analogs with such replacements as D-Ser, Ala, Thr, and Ac-Ser have been synthesized. These analogs were evaluated for their ability to hydrolyze the phosphoinositide in human neuroblastoma SH-EP cells. It was found that the α-amino group and L-configuration of Ser-1 are very important in the activation of receptors. N-Acetylation or deletion of Ser-1 completely eliminated the activity of SFLLRNP (a half-maximal effective concentration, EC50 = 0.89 μM (1 M = 1 mol dm-3)), and these modifications induced no antagonist activity. Incorporation of D-Ser also drastically diminished the activity, but retained about 50% activity of the maximal response by 100 μM SFLLRNP. The Ser/Ala substitution sustained 30% of the activity of SFLLRNP to elicit a full stimulation. The Ser/Thr substitution, however, enhanced the activity (20%) in spite of its decreased activity (60%) reported for platelet aggregation. These results indicated that the β-hydroxyl group of Ser-1 is important to receptor activation, but not essential. The effect of chemical modifications on the receptor activities of the tethered ligand is discussed with regard to the efficacy between phosphoinositide hydrolysis and biological activities..
115. Hideki Sumimoto, Yohko Kage, Hiroyuki Nunoi, Hiroyuki Sasaki, Takeru Nose, Yasuyuki Fukumaki, Motonori Ohno, Shigeki Minakami, Koichiro Takeshige, Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.91.12.5345, 91, 12, 5345-5349, 1994.06, The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91(phox)) and 22-kDa (p22(phox)) subunits, and two cytosolic factors (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47(phox) (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C- terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22(phox) and p67(phox) but fails to interact with a mutant p22(phox) carrying a Pro-156 → Gln substitution in a proline- rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins..
116. Kensaku Mizuno, Hayao Inoue, Michio Hagiya, Shin Shimizu, Takeru Nose, Yasuyuki Shimohigashi, Toshikazu Nakamura, Hairpin loop and second kringle domain are essential sites for heparin binding and biological activity of hepatocyte growth factor, Journal of Biological Chemistry, 269, 2, 1131-1136, 1994.01, Hepatocyte growth factor (HGF) has a strong affinity for heparin. About one fourth of HGF secreted from MRC-5 human embryonic lung fibroblast cells was found to be associated with heparin and heparan sulfate proteoglycan on the cell surface and extracellular matrix. To identify heparin-binding sites within the HGF molecule, we constructed variously deleted mutant HGFs and examined their binding ability to an immobilized heparin column. Native HGF and mutant HGFs, including d-K1 (deletion of the first kringle domain), d- K3 (deletion of the third kringle domain), d-K4 (deletion of the fourth kringle domain), d-β (deletion of β-chain), and HK1K2 (consisting of the N-terminal hairpin loop and the first two kringle domains), tightly bound to a heparin column, but d-H (deletion of the N-terminal hairpin loop) and d- K2 (deletion of the second kringle domain) markedly decreased binding ability to the column. These observations suggest that the N-terminal hairpin loop and the second kringle domain are essential for the heparin-binding of HGF. The finding that HK1K2 competed the binding of 125I-HGF to immobilized heparin provided additional evidence that the N-terminal half of HGF α-chain is the principal heparin-binding site. The hairpin loop in HGF possesses a cluster of basic amino acid residues and a highly positive net charge, when compared with hairpin loop structures in the other proteins, plasminogen and HGF-like protein. The second kringle domain in HGF has the basic amino acid cluster in the central region. Thus, it is likely that the basic clusters in these domains cooperatively contribute to the binding of HGF to the anionic heparin or heparan sulfate molecule..
117. Takeru Nose, Yasuyuki Shimohigashi, Shosaku Hattori, Hiroshi Kihara, Motonori Ohno, Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurs okinavensis (himehabu snake) venom which releases fibrinopeptides A and B, Toxicon, 10.1016/0041-0101(94)90309-3, 32, 12, 1509-1520, 1994.01, A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavensis (himehabu snake) venom, releases specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for α-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI=8.1) than okinaxobin I (pI=5.4). The N-terminal sequence was highly similar to those of okinazobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methly ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethly ketone, indicating that the enzyme is a serine protease like α-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to α-thrombin..
118. Toyoka Fukagawa, Takeru Nose, Yasuyuki Shimohigashi, Tomohisa Ogawa, Naoko Oda, Kin ichi Nakashima, Chun Chang Chang, Motonori Ohno, Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus Gramineus (green habu snake) venom, Toxicon, 10.1016/0041-0101(93)90255-H, 31, 8, 957-967, 1993.08, T. Fukagawa, T. Nose, Y. Shimohigashi, T. Ogawa, N. Oda, K. Nakashima, C.-C. Chang and M. Ohno. Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon 31, 957-967, 1993.-Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca2+-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3-5.2 × 10-4 M) for Ca2+ between these PLA2s and Tyr-28-containing PLA2s. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2s to Ca2+..
119. Hiroshi Sakamoto, Yasuyuki Shimohigashi, Iori Maeda, Takeru Nose, Kin‐ichi ‐i Nakashima, Ichiro Nakamura, Tomoshisa Ogawa, Motonori Ohno, Keiichi Kawano, Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions, Journal of Molecular Recognition, 10.1002/jmr.300060207, 6, 2, 95-100, 1993.06, A complete series of configurationally isomers (L‐L, L‐D, D‐L AND D‐D) of a dipeptide Leu‐Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz 1H NMR, the L‐D and D‐L isomers, but not hte L‐L and D‐D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu‐βCH2 and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe‐βCH2, the NOE enhancement observed between Leu‐δCH3 and Phe‐phenyl groups revealed that these groups are in close proximity. These data indicated that L‐D and D‐L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac‐Try‐OEt as a substrate, the L‐L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D‐L isomer was strongest with Ki of 2.2 × 10−5 M. It was found that the D‐L isomer was only slowly hydrolysed but the L(or D)‐D isomer was not. H‐D‐Phe‐L‐Leu‐OBzl with the inverse sequence of H‐D‐Leu‐L‐Pre‐OBzl inhibited chymotrypsin more strongly (Ki = 6.3 × 10−6 M). Since the free acid analogue of the D‐L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester‐benzyl group fits the S1 site of chymotrypsin, while the side chain‐side chain complexing hydrophobic core fits the S2 site..
120. Takeru Nose, Yasuyuki Shimohigashi, Motonori Ohno, Tommaso Costa, Naokata Shimizu, Yoshio Ogino, Enhancement of thrombin receptor activation by thrombin receptor-derived heptapeptide with para-fluorophenylalanine in place of phenylalanine, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1993.1680, 193, 2, 694-699, 1993.06, Thrombin receptor-derived peptide SFLLRNP (one-letter amino acid code) which corresponds to the N-terminal heptapeptide of tethered ligand is able to activate thrombin receptor and to stimulate the phosphoinositide (PI) turnover. The replacement of Phe-2 by Ala eliminated this activity completely, showing the crucial role of the Phephenyl group in receptor activation. It was found that substitution of parafluorophenylalanine ((p- F)Phe) for Phe-2 enhanced several times the PI-turnover activity of SFLLRNP. This is the first example to date of a substitution with one order of magnitude greater increase in receptor activation. The Phe-2/Tyr substitution diminished the activity drastically (almost 2% of SFLLRNP), indicating the importance of hydrophobicity of Phe2-phenyl. The Phe-2/Leu substitution, however, diminished also the activity (less than 2% of SFLLRNP). These results suggested that highly specific hydrophobic interaction exists between Phe-2 of the tethered ligand and its binding site in thrombin receptor..
121. Akinori Nagatomo, Kohta Sakai, Takeru Nose, Yasuyuki Shimohigashi, Motonori Ohno, Occurrence of an allosteric transition in the modification of papain withl-1-acetyl-2,3-dihydropyrrolo[2,3-b]-indole-2-carboxamide, Journal of Chromatography A, 10.1016/0021-9673(92)80138-K, 597, 1-2, 411-413, 1992.04, When papain was reacted with l-1-acetyl-2,3-dihydropyrrolo[2,3-b]indole-2-carboxamide at pH 8.0, inactivation occurred accompanied by modification of Cys-25 in the active site. Plots of pseudo first-order rate constants against the reagent concentrations yielded an anomalous sigmoidal curve, suggesting that papain responded to this reagent in an allosteric manner. This is supported by the fact that the presence of a moderate concentration (a twenty-fold molar excess) of Nα-acetyl-l-tryptophanamide over papain accelerated the inactivation..