• Korean Journal of Food Science and Technology
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• v.46 no.6
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• pp.716-722
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• 2014

Aminopeptidase-active fractions from crude extract of the hepatopancreas of a common squid (Todarodes pacificus) were obtained using acetone (AC; 30-40%) and ammonium sulfate precipitation (AS; 60-70% saturation), anion exchange (AE-II; 0.2 M NaCl) and gel filtration chromatography (GF-I; 30-50 kDa), respectively. The debittering capacity of GF-I fraction based on the aminopeptidase activity (89.2 U/mg), recovery (56.6%) and sensory evaluation (1.0) was better than that of other fractions. Release of amino acids increased as incubation time was increased, and the bitterness of the enzyme reaction mixtures decreased. Incubation with the GF-I fraction for 24 h resulted in the hydrolysis of several peptides, as revealed by reverse-phase HPLC profiles. Peaks 3, 5 and 6 showed the decreased area (%), whereas peaks 1, 2 and 4 showed the increased area. The GF-I fractions were found to be suitable for reducing bitterness in protein hydrolysates by catalyzing the hydrolysis of bitter peptides.

• 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.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.959-963
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• 2015

Purpose: To find parameters that can increase alpha-fetoprotein (AFP) sensitivity and so help in accurate diagnosis and rapid management of hepatocullular carcinoma (HCC), as AFP has limited utility of distinguishing HCC from benign hepatic disorders for its high false-positive and false negative rates. Materials and Methods: Serum levels of AFP, 5'-nucleotidase enzyme activity (5-NU) and leucine aminopeptidase enzyme (LAP) activity were measured in 40 individuals. Results: LAP and 5'NU were elevated in HCC at p<0.001. Pearson correlation coefficients showed that changes in AFP exhibited positive correlation with both 5'-NU and LAP at (p<0.001). The complementary use of LAP only with AFP resulted in an increase in sensitivity of AFP from 75% to 90% in detecting HCC. The complementary use of both LAP and 5-NU with AFP resulted in an increased sensitivity of AFP in detecting HCC from 75% to 95%. Conclusions: LAP and 5-FU can be determined in HCC patients in combination with AFP to improve its sensitivity and decrease false negative results.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.6
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• pp.849-860
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• 2021

This study investigated the preparation of seasoned anchovy sauce (SAS) and its functional characteristics by using aminopeptidase active fractions (AAFs) derived from squid Todarodes pacificus hepatopancreas as a bitter taste improver. As the base of the SAS, a hydrolysate (AAAH) prepared by continuously treating raw anchovies with Alcalase-AAF was used. The high-performance liquid chromatography profile of the AAAH suggested that the action of AAFs decreased the hydrophobicity of the N-terminal peptide related to bitterness in the protein hydrolysates. SAS was prepared by blending with the AAAH and other ingredients. The crude protein (2.5%), carbohydrates (18.4%), amino acid-nitrogen (1,325.1 mg/100 mL), and total free and released amino acids (FRAAs, 700.2 mg/100 mL) of SAS were higher than those of commercial anchovy sauce (CAS). Sensory evaluation revealed that SAS was superior to CAS in flavor, color, and taste. The main FRAAs of SAS were glycine (16.8%), alanine (13.2%), glutamic acid (7.8%), and leucine (7.3%). The amino acids that had a major influence on the taste according to the SAS taste values were glutamic acid, aspartic acid, alanine, and histidine. The angiotensin-converting enzyme inhibitory (2.21 mg/mL) and antioxidant activities (3.58 mg/mL) of SAS were superior to those of CAS.

• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.530-536
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• 2009

Cobalt chloride ($CoCl_2$) treatment of cells in vitro has been shown to induce cellular changes that are similar to those seen following hypoxia. To identify genes that are differentially expressed in response to treatment with $CoCl_2$, we compared the mRNA expression profiles of PC-3 cells that were treated with $CoCl_2$ with those of untreated PC-3 cells, using specific arbitrary primers and two anchored oligo(dT) primers provided in the ACP-based GeneFishing kits. The results of this study demonstrated that the puromycin-sensitive aminopeptidase (PSA) gene was down regulated in PC-3 cells that were treated with $CoCl_2$. This downregulation of PSA expression, in turn, suppressed the proliferation, migration, and invasion of PC-3 cells, as well as the secretion and expression of matrix metalloproteinase-9 (MMP-9).

• The Korean Journal of Soil Zoology
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• v.3 no.2
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• pp.51-57
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• 1998

A new rod-shaped endospore-forming bacterium isolated from the hindgut flora of the termite, Reticulitermes speratus kyushuenesis Morimoto is described. The isolate stained Gram positive, but the KOH test and the test for L-alanine aminopeptidase were negative. The length of a single cell varies from 2.5-9.0 $\mu $m, and the cell is about 0.5-0.7$\mu $m thick. The isolate had a high cellulolytic ability and was identified as Bacillus amyloliquefaciens.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• KSBB Journal
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• v.10 no.3
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• pp.271-282
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• 1995

The authentic hGH converted from met-hGH by immobilized ApM was purified by successive chromatographic processes based on the differences in isoelectric points, hydrophobicities and charges. The final recovery yield was about 14.1% and the specific activity of the purified hGH was 2.75IU per mg when assayed by enzyme immunoassay. The purified hGH was verified to be authentic hGH through the analysis of amino acid composition, amino-terminal amino acid sequence, carboxy-terminal amino acid and tryptic peptide map. The purity of purified hGH was higher than that of commercial hGH when assessed by SDS-PAGE, PAGE, IEF and HSGF. In weight-gain assay and tibia test with hypophysectomized rats, the hGH produced in this study showed the same growth effect as the commercial hGH.

• BMB Reports
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• v.45 no.6
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• pp.360-364
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• 2012

Uptake of circulating glucose into the cells happens via the insulin-mediated signalling pathway, which translocates the glucose transporter 4 (GLUT4) vesicles from the intracellular compartment to the plasma membrane. Rab GTPases are involved in this vesicle trafficking, where Rab GTPases-activating proteins (RabGAP) enhance the GTP to GDP hydrolysis. TBC1D4 (AS160) and TBC1D1 are functional RabGAPs in the adipocytes and the skeletonal myocytes, respectively. These proteins contain two phosphotyrosine-binding domains (PTBs) at the amino-terminus of the catalytic RabGAP domain. The second PTB has been shown to interact with the cytoplasmic region of the insulin-regulated aminopeptidase (IRAP) of the GLUT4 vesicle. In this study, we quantitatively measured the ${\sim}{\mu}M$ affinity ($K_D$) between TBC1D4 PTB and IRAP using isothermal titration calorimetry, and further showed that IRAP residues 1-49 are the major region mediating this interaction. We also demonstrated that the IRAP residues 1-15 are necessary but not sufficient for the PTB interaction.

• Journal of Veterinary Clinics
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• v.8 no.1
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• pp.31-45
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• 1991

Present experiment was performed in order to establish the optimal reaction conditions for determination of urinary AAP and GRS activities and to investigate the applicability of urinary AAP and GRS in nephrotoxicity test in rat. The results were as follows ; 1. The optimal pH of phosphate buffer for determination of urinary AAP activity was 7.8. 2. The Michaelis constant of urinary AAP ranged from 0.8 to 1.0mmol/$\ell$ 3. The optimal wave length for determination of urinary GRS activity was 405nm. 4. The optimal pH of acetate buffer for determination of urinary GRS activity was 5.6. 5. The Michaelis constant of urinary GRS ranged from 0.65~0.79mmo1/$\ell$. 6. The AAP activities in gel-filtered samples were significantly higher than those in crude samples. Mean values of AAP activities in gel-filtered samples and crude samples were 29$\pm$20 and 20$\pm$13U/$\ell$, respectively. 7. There was not significant difference between gel-filtered samples and crude samples in urinary GRS activities. Mean values of GRS activities in gel-filtered samples and crude samples were 57$\pm$40 and 56$\pm$39U/$\ell$, respectively. 8. Limits of linearity of urinary AAP and GRS activities were 2.0 and 3.6U/$\ell$, respectively. 9. Within-run imprecisions of the assays, were acceptable, as the coefficients of the AAP activities ranged from 5.5 to 6.3% and those of GRS activities ranged from 1.4 to 6.2%, respectively. 10. Urinary AAP excretion was 675$\pm$227mu/24hrs.kg before administration of potassium dichromate, and increased significantly to 4246$\pm$2567mU/24hrs.kg within 24 hours after administration of potassium dichromate. 11. Urinary GRS excretion did not increase significantly after administration of potassim dichromate. 12. From these findings it is concluded that urinary AAP excretion is early and sensitive Indicator to detect kidney damage in nephrotoxicity experiment.