• Applied Biological Chemistry
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• v.41 no.8
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• pp.608-610
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• 1998

To investigate the inhibitory activity of 2-hydroxy-3-amino-4-phenylbutyrate-harboring aminopeptidase N inhibitors, p-nitro-AHPA-peptide derivatives (1 and 2) and an AHPA-peptide derivative (3) were synthesized by chain elongation from C-terminal end using DCC/HOBt as a coupling reagent. The peptides $1{\sim}3$ exerted strong inhibitory activities against aminopeptidase N with $IC_{50}$ values of 1.8, 7.3 and $24.0\;{\mu}g/ml$, respectively, and cytotoxicity on cancer cell lines in vitro.

• Applied Biological Chemistry
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• v.38 no.2
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• pp.100-105
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• 1995

Media optimization for the production of MR-387A and B, novel aminopeptidase M inhibitors by Streptomyces sp. SL-387 isolated from a soil was studied. Optimized medium was consisted of 1% glucose, 3% soybean meal, 0.2% yeast extract, 0.1% beef extract, 0.3% NaCl, 0.01% $K_2HPO_4$, 0.3% $CaCO_3$, 0.001% $MnCl_2{\cdot}4H_2O$, 0.001% $ZnCl_2{\cdot}7H_2O$, and 0.0005% $MgSO_4{\cdot}7H_2O$, and adjusted to pH 7.0 before autoclaving. When the optimized medium was used as a fermentation medium, maximum productivity of MR-387 was reached at 120 hours of fermentation, and total productivity was 909.1 U/ml.

• Applied Biological Chemistry
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• v.38 no.3
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• pp.196-201
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• 1995

Chemo- and numerical taxonomic studies on the isolate SL-387 producing novel aminopeptidase M inhibitors MR-387A and B were carried out The genus of the isolate was determined as Streptomyces by cultural and morphological data and chemical indices. Forty one taxonomic unit characters were tested for determining the species of the isolate, and the data were analyzed numerically using a computer program as called TAXON. The isolate was best matched to Streptomyces neyagawaensis in the major cluster 18 of Streptomyces with $S_{SM}$ value of 75.67%. On the base of chemotaxonomic data and TAXON analysis, the isolate SL-387 was identified to be a member of Streptomyces neyagawaensis.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1629-1633
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• 2015

Peptide assimilation in Helicobacter pylori necessitates a coordinated working of the peptide transport systems (PepTs) and aminopeptidase (PepA). We found that H. pylori hydrolyzes two detector peptides, _L-phenylalanyl- _L-3-thiaphenylalanine (PSP) and _L-phenylalanyl- _L-2-sulfanilylglycine (PSG), primarily before intake and excludes their antibacterial effects, whereas Escherichia coli readily transports them with resultant growth inhibition. PSP assimilation by H. pylori was inhibited by aminopeptidase inhibitor bestatin, but not by dialanine or cyanide-m-chlorophenylhydrazone, contrary to that of E. coli. RT- and qRT-PCR analyses showed that H. pylori may express first the PepTs (e.g., DppA and DppB) and then PepA. In addition, western blot analysis of PepA suggested that the bacterium secretes PepA in response to specific inducers.

• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.110-110
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• 2005

• Applied Chemistry for Engineering
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• v.26 no.1
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• pp.111-115
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• 2015

Two new analogs of bestatin were prepared from $\small{D}$-leucine and $\small{D}$-valine in a stereoselective and efficient way. An aminopeptidase inhibitor bestatin shows significant biological effects on immunomodulation and is marketed for the treatment of acute myelocytic leukemia. The key intermediates, trans-oxazolidine methyl esters 2a and 2b, were obtained with more than 20 to 1 stereoselectivity in a one-pot procedure by the three cascade reactions between N-hydroxymethyl protected ${\alpha}$-amino aldehydes (4a and 4b) and phenylsulfonylnitromethane ($PhSO_2CH_2NO_2$) and the following in-situ ozonolysis. Basic hydrolysis of 2a and 2b, and then the peptide coupling with $\small{L}$-Leu-OMe produced the protected derivatives of two new bestatin analogs, 3a and 3b, respectively. The new isobutyl and isopropyl analogs of bestatin (1a and 1b) were produced in overall 51% and 38% yields, respectively, with high stereoselectivity from the corresponding protected ${\alpha}$-amino aldehydes 4 in a six-step process.

• BMB Reports
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• v.36 no.2
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• pp.214-222
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• 2003

A lectin named AAL has been purified from the fruiting bodies of the edible mushroom Agrocybe aegerita. AAL consisted of two identical subunits of 15.8 kDa, its pI was about 3.8 determined by isoelectric focusing, and no carbohydrate was discerned. Being treated by pyrogultamate aminopeptidase, the blocked N-terminus of AAL was sequenced as QGVNIYNI. AAL agglutinated human and animal erythrocytes regardless of blood type or animal species. Its hemagglutinating activity was unaffected by acid or alkali treatment and demetalization or addition of divalent metals $Mg^{2+}$, $Ca^{2+}$ and $Zn^{2+}$. AAL was toxic to mice: its LD50 was 15.85 mg per kilogram body weight by intraperitoneal injection. In this study, two novel activities of AAL were proved. It showed inhibition activity to infection of tobacco mosaic virus on Nicotiana glutinosa. The result of IEF suggested that AAL attached to TMV particles. Mycelia differentiation promotion was the other interesting activity. AAL promoted the differentiation of fruit body primordia from the mycelia of Agrocybe aegerita and Auricularia polytricha. AAL antiserum was prepared and immunologically cross-reactived with several proteins from five other kinds of mushrooms. These results suggested that AAL probably was a representative of a large protein family, which plays important physiological roles in mushroom.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.952-959
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• 2007

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multimers. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3mg/l and analyzed by matrixassisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.

• Journal of Pharmaceutical Investigation
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• v.26 no.3
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• pp.175-185
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• 1996

To inhibit the enzymatic degradation of leucine enkephalin (Leu-Enk) and its synthetic analog. $[D-ala^2]$-leucine enkephalinamide (YAGFL), in the nasal, rectal and vaginal mucosal and serosal extracts of rabbits, effects of enzyme inhibitors such as amastatin (AM), puromycin (PM), thiorphan (TP), thimerosal (TM), EDTA, N-carboxymethyl-Phe-Leu (CPL), phenylethyl alcohol (PEA), phenylmercuric acetate (PMA), benzalkonium chloride (BC) and modified cyclodextrins, alone or in combination, were observed by assaying the pentapeptides staying intact during incubation. Mucosa extracts were prepared by exposing freshly-excised mucosal specimens mounted on Valia-Chien cells to isotonic phosphate buffer while stirring. The degradation of Leu-Enk and YAGFL followed the apparent first-order kinetics. The half-lives (mean) in the nasal, rectal and vaginal mucosal extracts were found to be 1.07, 0.33 and 1.14 hr for Leu-Enk, and 16.9, 6.2 and 6.8 hr for YAGFL, respectively. AM or PM, which is an aminopeptidase inhibitor, did not show a sufficient inhibition of Leu-Enk $(50\;{\mu}g/ml)$ degradation in all kinds of extracts. $Dimethyl-{\beta}-cyclodextrin\;(DM-{\beta}-CyD)$ decreased the degradation rate constants of Leu-Enk about 2 or 3 times, comparing with no additive. However, the use of mixed inhibitors of AM $(50\;{\mu}M)$/TM (0.25 mM)/EDTA (5 mM) resulted in a full stabilization of Leu-Enk by decreasing the degradation rate constants 67.3, 161.3 and 113.8 times far the nasal, rectal and vaginal mucosal extracts, respectively, comparing with no inhibitor. With mixed inhibitors, Leu-Enk remained intact more than 90% after 6 hr-incubation. In the stabilization of YAGFL, hM, TP or CPL alone showed little efffct, and some additives demonstrated a considerable inhibition of YAGFL degradation in the rank order of TM > BC > EDTA. However, the addition of mixed inhibitors such as TM (0.5 mM) and EDTA (5 mM) into the extracts protected YAGFL from the degradation by more than 85% even after 24 hr-incubation, suggesting almost complete inhibition of YAGFL degradation in the extract. On the other hand, $DM-{\beta}-CyD\;or\;hydroxypropyl-{\beta}-cyclodextrin$ (10%) were also found to retard enzymatic degradation rates of YAGFL markedly, and resulted in staying intact more than 80% of YAGFL in the nasal and vaginal mucosal extracts, and more than 60% in the rectal mucosal extract after 16 hr-incubation.

• Parasites, Hosts and Diseases
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• v.35 no.1
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• pp.39-46
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• 1997

The present study was undertaken to determine whether live T. uaginnlis degrades human secretory IgA, serum If and IgG molecules. Human immunoglobulins were exposed to live trophozoites, parasite Iysate, and excretory-secretory product (ESP) of T ucginnlis. To determine the fragmentation of immunoglobulins, the reaction sample was subjected to SDS-PAGE and EITB, and peroxidase conjugated antihuman IgA and IgG were used as probes. Live trophozoites degraded secretory IgA, serum IgA and IgG, and degradation were pressed forward by the prolongation of the incubation time and by increasing the number of trichomonads respectively. Also the Iysates and ESP of trichomonads degraded IgA and IgG. The cysteine and serine proteinase inhibitors such as I-64, antipain, iodoacetic acid, iodoacetamide, TLCK reduced the ability of cleaving immunoglobulins. The proteinase activity and cytotoxicity of T. uaginnlis to HeLa cells were decreased when live T. vusinalis was treated with metallo-proteinase inhibitor as well as cysteine and serine proteinase inhibitors. These results suggest that proteinase secreted from live T ucginclis may play a part role in host pathogenesis by T. uosinnlis, and the cleaving ability of host immunoglobulins by the proteinase may contribute as a one of immune evasion mechanism for parasite survival in the host.