• IMMUNE NETWORK
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• v.13 no.3
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• pp.86-93
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• 2013

In the present study, we investigated if priming of autoreactive $CD8^+T$ cells would be inhibited by competitive peptides for major histocompatibility complex (MHC) class I binding. We used a mouse model of vitiligo which is induced by immunization of $K^b$-binding tyrosinase-related protein 2 (TRP2)-180 peptide. Competitive peptides for $K^b$ binding inhibited IFN-${\gamma}$production and proliferation of TRP2-180-specific $CD8^+T$ cells upon ex vivo peptide restimulation, while other MHC class I-binding peptides did not. In mice, the capability of inhibition was influenced by T-cell immunogenicity of the competitive peptides. The competitive peptide with a high T-cell immunogenicity efficiently inhibited priming of TRP2-180-specific $CD8^+T$ cells in vivo, whereas the competitive peptide with a low T-cell immunogenicity did not. Taken together, the inhibition of priming of autoreactive $CD8^+T$ cells depends on not only competition of peptides for MHC class I binding but also competitive peptide-specific $CD8^+T$ cells, suggesting that clonal expansion of autoreactive T cells would be affected by expansion of competitive peptide-specific T cells. This result provides new insights into the development of competitive peptides-based therapy for the treatment of autoimmune diseases.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.727-731
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• 2005

Synthesized Ile-Ala-Val-Pro (IAVP) peptide, which has the highest hypocholesterolemic effect among a number of synthesized derivatives of Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA) isolated from 11S globulin of soy protein by pepsin digestion, was selected for investigation in the present study. Using a recombinant Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), we studied in detail the inhibition of this enzyme by IAVP and compared the action of this peptide to that of lovastatin, a known competitive inhibitor of this enzyme. The concentration of IAVP required for 50% inhibition ($IC_{50}$) of HMGR activity in given experimental conditions was $340\;{\mu}M$. Kinetic analysis revealed that the studied peptide is a competitive inhibitor of HMGR with respect to both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and nicotinamide adenine dinucleotide phosphate (NADPH), with an equilibrium constant of inhibitor binding ($K_i\;=\;[E][I]/[EI]$) of $61{\pm}1.2\;{\mu}M$ and $157{\pm}4.4\;{\mu}M$, respectively. At the same conditions, $K_i$ and $IC_{50}$ for lovastatin were $2.2{\pm}0.1\;nM$ and 12.5 nM, respectively. Thus, the given peptide interacts with HMGR as a bisubstrate, consequently blocking access of both substrates to the active sites. The achieved results suggest the design of new peptide sequences having a higher relative affinity to binding sites of this enzyme and an enhancement of their hypocholesterolemic properties.

• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.92-97
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• 1996

Portage kinetic constants of peptide transport can be measured by competitive spectrophotometry. The kinetic constants of L-Glu-L-Glu transport in Escherichia coli were ascertained using L-Phe-L-3-thia-Phe (PSP) as a detector. Since the production of thiophenol upon intracellular hydrolysis of PSP was competitively inhibited by L-Glu-L-Glu, it was able to compute the kinetic constants of L-Glu-L-Glu using this method. The resulted data were in agreement with the values obtained by the method of Michaelis-Menten kinetics. The potential of this method was examined against dipeptide transport systems in various microorganisms. These results strongly suggest that the overall properties of individual systems for dipeptide transports can be easily characterized by competitive spectrophotometry.

• IMMUNE NETWORK
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• v.10 no.3
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• pp.99-103
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• 2010

Background: Glycogen synthase kinase $3{\beta}$ ($GSK3{\beta}$) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological $GSK3{\beta}$ inhibitors in rodent models of septic shock. Since most of the $GSK3{\beta}$ inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive $GSK3{\beta}$ inhibitors is needed. Methods: Based on the unique phosphorylation motif of $GSK3{\beta}$, we designed and generated a novel class of $GSK3{\beta}$ inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival. Results: We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock. Conclusion: Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.899-902
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• 2005

Peptide assimilation by Helicobacter pylori was investigated using L-phenylalanyl-3-thia-phenylalanine (PSP) as a detector peptide; the release of thiophenol upon enzymatic hydrolysis of PSP was spectrophotometrically detected with the aid of 5,5'-dithiobis[2-nitrobenzoic acid] (DTNB). By adding PSP to whole-cell suspension, thiophenol was produced progressively, resembling that found in Esherichia coli or Staphylococcus aureus. Interestingly, the rate of thiophenol production by H pylori in particular was markedly reduced when cells were pretreated with trypsin, indicating surface exhibition of peptidase. According to the competitive spectrophotometry using alanyl-peptides, H pylori did not appear to assimilate PSP through the peptide transport system. No discernible PSP assimilation could be ascertained in H pylori cells, unless provided with some additives necessary for peptidase activity, such as $Ni^{2+}\;or\;Mg^{2+}$ and an appropriate concentration of potassium or ammonium salts. These observations strongly suggest that, regardless of a presumptive peptide transport system, peptide assimilation of H. plori appears to be highly dependent upon milieu conditions, due to unique peptidase exhibition on the cell surface.

• Mass Spectrometry Letters
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• v.4 no.4
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• pp.71-74
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• 2013

In the present study, we report that the charge-directed (assisted) peptide dissociation products, such as b- and y-type peptide backbone fragments, were the major products in MS/MS and $MS^3$ applications of some o-TEMPO-Bz-C(O)-peptide ions, while radical-driven dissociation products, such as a/x and c/z-type fragments, were previously shown to be the major products in the free radical initiated peptide sequencing mass spectrometry (FRIPS MS). Those o-TEMPO-Bz-C(O)-peptides share a common feature in their sequences, that is, the peptides do not include an arginine residue that has the highest proton affinity among free amino acids. The appearance of b- and y-type fragments as major products in FRIPS MS can be understood in terms of the so-called "mobile-proton model". When the proton is highly mobilized by the absence of arginine, the chare-directed peptide dissociation pathways appear to be more competitive than the radical-driven dissociation pathways, in our FRIPS experiments.

• Fisheries and Aquatic Sciences
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• v.21 no.5
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• pp.13.1-13.8
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• 2018

Amyloid plaque, also called senile plaque, the product of aggregation of ${\beta}$-amyloid peptides ($A{\beta}$), is observed in brains of the patients with Alzheimer's disease (AD) and is one of the key factors in etiology of the disease. In this study, hydrolysates obtained from the sea hare (Aplysia kurodai) were investigated for ${\beta}$-secretase inhibitory peptide. The sea hare's muscle protein was hydrolyzed using six enzymes in a batch reactor. Trypsin hydrolysate had highest ${\beta}$-secretase inhibitory activity compared to the other hydrolysates. ${\beta}$-secretase inhibitory peptide was separated using Sephadex G-25 column chromatography and high-performance liquid chromatography on a C18 column. ${\beta}$-secretase inhibitory peptide was identified as eight amino acid residues of Val-Ala-Ala-Leu-Met-Leu-Phe-Asn by N-terminal amino acid sequence analysis. $IC_{50}$ value of purified ${\beta}$-secretase inhibitory peptide was $74.25{\mu}M$, and Lineweaver-Burk plots suggested that the peptide purified from sea hare muscle protein acts as a competitive inhibitor against ${\beta}$-secretase. Results of this study suggest that peptides derived from sea hare muscle may be beneficial as anti-dementia compounds in functional foods or as pharmaceuticals.

• Fisheries and Aquatic Sciences
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• v.15 no.3
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• pp.183-190
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• 2012

The purpose of this study was the purification and characterization of an angiotensin I converting enzyme (ACE) inhibitory peptide purified from enzymatic hydrolysates of rainbow trout Oncorhynchus mykiss muscle. After removal of lipid, the approximate composition analysis of the rainbow trout revealed 24.4%, 1.7%, and 68.3% for protein, lipid, and moisture, respectively. Among six hydrolysates, the peptic hydrolysate exhibited the highest ACE inhibitory activity. We attempted to purify ACE inhibitory peptides from peptic hydrolysate using high performance liquid chromatography on an ODS column. The $IC_{50}$ value of purified ACE inhibitory peptide was $63.9{\mu}M$. The amino acid sequence of the peptide was identified as Lys-Val-Asn-Gly-Pro-Ala-Met-Ser-Pro-Asn-Ala-Asn, with a molecular weight of 1,220 Da, and the Lineweaver-Burk plots suggested that they act as a competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides purified from rainbow trout muscle protein may be beneficial as anti-hypertension compounds in functional foods.

• BMB Reports
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• v.31 no.1
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• pp.58-63
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• 1998

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41(isotype IgG1) was identified, whose specific reactivity against gp41(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptied. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp35, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.

• Journal of Radiation Industry
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• v.7 no.2_3
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• pp.127-134
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• 2013

Bombesin receptors are overexpressed in many kinds of human tumors. In particular, the gastrin releasing peptide receptor (GRPR) which is also called bombesin receptor subtype 2, has been identified in prostate cancer. In the present study, we developed a bombesin antagonist-based $^{177}Lu$-labeled peptide, $^{177}Lu$-DOTA-$Ala(SO_3H)$-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-$NH_2$ (DOTA-sBBNA). DOTA-sBBNA was prepared using a solid phase synthesis method. It was labeled with $^{177}Lu$ by a high radiolabeling yield (>98%), and its Log P value was -2.05. The radiolabeled peptide was highly stable in serum incubation at $37^{\circ}C$ for 48 hr. A competitive displacement of $^{125}I-[Tyr^4]$-Bombesin on the PC-3 human prostate carcinoma cells revealed that the $IC_{50}$ value of the peptide was 6.76 nM indicating a highly nanomolar binding affinity for GRPR. These results suggest that $^{177}Lu$-DOTA-sBBNA can be a potential candidate for targeting prostate cancer, and further studies to evaluate its biological characteristics are needed.