• Applied Biological Chemistry
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• v.31 no.4
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• pp.356-360
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• 1988

The alkaline protease producing bacteria isolated from soil and identified as Bacillus subtilis. The optimum medium for alkaline protease production from the microorganism was as follows; soluble starch, 1.5% ; proteose peptone, 0.5% ; $K_2HPO_4$, 0.1% ; $MgSO_4{\cdot}7H_2O$, 0.02% and sodium carbonate, 1.0%. The optimum temperature for alkaline protease production was $35^{\circ}C$, and the initial pH of medium was pH 10.5. The alkaline protease activity was about 2,300 U per ml of culture broth by Casein-Folin Method. A 9.2 fold purification of alkaline protease was obtained from culture broth. The recovery was 14% and purified enzyme was identified as single band, and its molecular weight was about 19,000. The optimum temperature for enzyme reaction was $70^{\circ}C$, and optimum pH was 12. The activity of purified enzyme was inhibited by metal ion ($Fe^{++}$), and Phenylmethylsulfonyl Fluoride, a serine protease inhibitor.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.804-807
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• 2006

Bacterial serine proteases, especially those from Bacillus, have been extensively studied. Intracellular serine protease 1 (Isp1) is responsible for most of the proteolytic activity in B. subtilis. To identify Isp1 substrates and study its physiological functions, a mutant of Isp1, which has lost the enzymatic activity, was constructed. Through a GST affinity chromatographic method, several Bacillus proteins that specifically interacted with S246A mutant Isp1 protein were isolated and then identified by MALDI-TOF analysis. ClpC and elongation factor Tu (EF-Tu) were among those proteins specifically bound to mutant Isp1. In addition, several proteins involved in stationary phase adaptive response (such as RNA polymerase sigma factor, spoIIIE) were also identified. These findings led us to suggest that the major function of this serine protease, whose expression is greatly increased during the stationary phase, is to mediate transition of the cell into the stationary phase in a proper and timely manner.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.2
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• pp.178-186
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• 2015

Protease inhibitors (PI) of trypsin and papain as target proteases from the roe of bastard halibut Paralichthys olivaceus were fractionated out using ammonium sulfate precipitation (A), DEAE 650M anion exchange chromatography (D), and Sephacryl S-300 gel filtration (S). The recovery percentages of the fractions with the strongest inhibitory activity for each fractionation method were 13% for the A4 fraction, 21.2% for the D3 fraction, and 21.3% for the S2 fraction, with specific inhibitory activities of the fractions toward trypsin and casein of 168, 139, and 218 U/mg, respectively, while no inhibition of papain was observed. The $IC_{50}$ for the trypsin-specific substrate $N{\alpha}$-benzoyl-$\small{L}$-arginine-p-nitroanilide (BAPNA) was 0.65, 1.55, 2.26, and 2.85 mg/mL for the A4, S2, A3, and D3 fractions, respectively. These results suggest that chromatographic fractionation methods (D and S) based on the molecular mass and charge of the protein were more effective at fractionating PI than was ammonium sulfate precipitation based on protein solubility, and that the bastard halibut roe extract acts as a serine protease inhibitor. Therefore, the PI fraction from fish roe might be useful for inhibiting proteases in foodstuffs, and could constitute an alternative food-grade inhibitor for the surimi industry.

• Molecular & Cellular Toxicology
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• v.2 no.2
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• pp.81-88
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• 2006

Sphingolipid metabolites regulate many aspects of cell proliferation, differentiation, and apoptosis. In the present study, we have assessed the effects of the novel phytosphingosine derivative, N-acetylphytospingosine (NAPS), on the depigmentation of murine B16F10 melanoma cells, and have also attempted to identify the possible signaling pathway involved, in comparison with $C_{2}-ceramide$. NAPS and $C_{2}-ceramide$ both inhibited the growth of the B16F10 cells in a dose-dependent manner. Melanin content and tyrosinase activity were significantly reduced in response to treatment with NAPS and $C_{2}-ceramide$ at concentrations in a range between $1-5\;{\mu}M$. However, the levels of tyrosinase mRNA, as well as the levels of tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2) genes and the level of tyrosinase protein remained unaffected by treatment with either NAPS or $C_{2}-ceramide$. We also attempted to determine the signaling pathway exploited by NAPS and $C_{2}-ceramide$. Interestingly, the phosphorylation of Akt/PKB at serine 473 by NAPS was reduced at the 5 minute mark, whereas $C_{2}-ceramide$ induced the phosphorylation of Akt/PKB at serine 473. Finally, Akt/PKB activity in the NAPS-treated cells was elevated in comparison with the untreated cells. LY294002, a specific PI3-K inhibitor which is located upstream of Akt/PKB, inhibited the phosphorylation of Akt/PKB, but induced an increase in melanin synthesis. These results suggest that the activation of Akt/PKB at serine 473 is related with the suppression of melanin production in the B16F10 mouse melanoma cells. Therefore, the mechanisms exploited by NAPS and $C_{2}-ceramide$ responsible for the depigmentation of B16F10 cells were concluded to involve the inhibition of melanosomal tyrosinase activity.

• Journal of Fisheries and Marine Sciences Education
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• v.26 no.4
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• pp.808-822
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• 2014

넙치 (Paralichthys olivaceus)로부터 elastase-like serine protease (PoElSp)를 암호화하는 cDNA를 클로닝하여 그 서열을 분석한 결과, PoElSp 유전자는 269 아미노산을 암호화하는 978염기쌍으로 구성되었다. PoElSp 유전자의 조직 특이적 발현 양상을 RT-PCR법으로 조사한 결과, 간, 비장 및 소장에서 그 발현이 크게 나타났다. lipopolysaccharide (LPS)로 인위적 세균감염을 유도한 후, 1시간째에 콩팥에서, 3시간째에는 근육에서, PoElSp 유전자의 발현이 크게 증가하였다. 또한, 이 유전자의 발현은 비장에서 LPS 주입 후 1-24시간동안 점차로 증가하였다. pro-mature PoElSp (proPoElSp)에 해당하는 cDNA를 pET32a 벡터 시스템을 이용하여 대장균에서 발현시켰다. 이 재조합 proPoElSp 단백질의 활성은 gelatin zymography 방법과 합성형광 Z-Phe-Arg-AMC의 분해법을 이용하여 측정하였다. 단백질 분해효소 활성을 위한 최적 pH는 7.5였다. 실험결과들을 종합하면, PoElSp 단백질은 면역 반응에서 중추적 역할을 하리라 판단된다.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5741-5745
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• 2013

Background: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value. Materials and Methods: This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status. Results: The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine. Conclusions: Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.

• Journal of the Korean Society of Food Science and Nutrition
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• v.5 no.1
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• pp.61-64
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• 1976

A survey of the free amino acids and organic acids in the shoot of Phyllostachys edulis was made by means of amino acid autoanalyzer and gas chromatograph. The results of the survey are summerized as follows. 1. Eighteen amino acids found in bamboo shoot were lysine, histidine, arginine, tryptophan, aspartic acid, glutamic acid, threonine, serine, proline, glycine, alanine, valine, leucine, isoleucine, methionine, tyrosine and phenylalanine, and an unknown was found. Serine showed the highest amount and more than about 44% of total free amino acids. 2. Oxalic acid was the major organic acid, and formic acid, acetic acid, maleic acid, succinic acid, fumaric acid, citric acid, tartaric acid and sorbic acid were determined, and two unknown were found.

• KSBB Journal
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• v.17 no.1
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• pp.49-53
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• 2002

The changes in the levels of $\gamma$ -aminobutyric acid (GABA) and some free amino acids were investigated in germinating brown rices. Ungerminated brown rices were germinated for 72 hrs by application of the following solutions: 1) distilled water, 2) 50 ppm lactic acid, 3) 5 mM glutamic acid. The GABA levels were enhanced in all germinated states of brown rices compared with ungerminated ones, highest in the germinated brown rices by 5 mM glutamic acid solution. Alanine levels were also enhanced significantly in the germinated brown rices. The levels of aspartic acid and glutamic acid were decreased significantly in all the germinated states. The levels of serine decreased during germination in the solutions of water and lactic acid were increased by the germination in the glutamic acid solution. The data show that germination of brown rices by the application of the glutamic acid solution can significantly increase the levels of GABA and can restore the serine level.

• BMB Reports
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• v.33 no.6
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• pp.493-498
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• 2000

Aquifex pyrophilus, a hyperthermophilic bacterium, has a serine-type protease that is located at the cell wall fraction with a mature size of 43 kDa. Molecular cloning of the protease gene revealed that it has an ORF of 619 amino acids with homologous catalytic site of serine-type proteases [Choi, I.-G., Bang, W.-K., Kim, S.-H., Yu, G. Y., J. Biol. Chem. (1999), Vol. 274, pp. 881-888]. Constructs containing different regions of the protease gene, including a alanine-substituted mutant at the active site serine, were constructed, and the factors affecting the expression level of the cloned protease gene in E. coli were examined. The presence of the C-terminus hydrophobic region of the protease hindered over-expression in E. coli. Also, the proteolytic activity of the expressed protein appeared to toxic to E. coli. An inactive form that deleted both of the N-terminal signal sequence and the C-terminal polar residues was over-expressed in a soluble form, purified to homogeneity, and its thermostability examined. The purified protein showed three disulfide bonds and three free sulfhydryl group. The thermal denaturation temperature of the protein was measured around $90^{\circ}C$ using a differential scanning calorimeter and circular dichroism spectrometry. The disulfide bonds were hardly reduced in the presence of reducing agents, suggesting that these disulfide bonds were located inside of the protein surface.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.34 no.2
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• pp.143-148
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• 2008

Variations of the seventeen amino acids(aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystein, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine) were analyzed in human hair sample by amino acid auto analyzer(AAA). The effect of bleaching and permanent wave manipulation on the amino acid composition of hair were investigated. Hair samples were collected from 10 males in their thirties. Hair samples were treated with 10 mL of 6 N hydrochloric acid at $110^{\circ}C$ for 24 h and analysed by AAA. The results showed that the amino acid content of normal hair(73.9%) decreased to 71.5% and 69.3% after bleaching and permanent wave treatment, respectively. Furthermore, mean contents of lysine and tyrosine in amino acids showed obviously decreased about 25% by permanent wave and bleaching treatment. On the other hand, serine, cystein, leucine and histidine were not changed.