• Applied Biological Chemistry
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• 제31권4호
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• pp.356-360
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• 1988

토양에서 분리된 Alkaline Protease생산균주는 Bacills subtilis로 동정되었다. 본 균주의 최적배양온도는 최적 배지조건에서 $35^{\circ}C$이며, 초기 배지 pH는 10.5였다. 효소역가는 Casein-Folin법으로 약 2,300 Unit/ml로 나타났다. 배양액에 일련의 정제과정을 실시한 결과 정제배수 9.2, 회수율 14%의 정제효소를 얻었으며, 분자량은 19,000이었다. 정제효소의 반응최적온도는 $70^{\circ}C$, 최적 PH는 12였다. $Fe^{++}$에 의해 활성이 저하되었으며, 또한 serine protease inhibitor인 PMSF에 의해 활성이 저해되었다. 이것으로 부터 본 효소는 활성부위에 serine을 갖는 serine protease의 일종임을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제16권5호
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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.

• 한국수산과학회지
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• 제48권2호
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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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• 제2권2호
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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.

• 수산해양교육연구
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• 제26권4호
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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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• 제14권10호
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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.

• 한국식품영양과학회지
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• 제5권1호
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• pp.61-64
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• 1976

P. edulis순(荀)의 free amino acid 및 organic acid의 종류(種類)와 함량(含量)을 측정(測定)한 결과(結果)는 다음과 같다. (1) Free amino acid는 18종(種)이 검출(檢出)되었는데, lysine histidine arginine tryptophan aspartic acid glutamic acid threonine serine proline glycine alanine valine methionine isoleucine leucine tyrosine phenylalanine 등 17종(種)이 정량(定量)되었으며 이들 중 serine이 가장 많았고, 1종(種)은 미확인(未確認)되었다. (2) Organic acid는 11종(種)이 검출(檢出)되었으며 이들 중 formic acid acetic acid oxalic acid maleic acid succinic acid fumaric acid citric acid tartaric acid sorbic acid 등 9종(種)이 확인(確認)되었고, 이들 중 oxalic acid가 가장 많아 291.14mg%이었으며, 2종(種)은 미확인(未確認)되었다.

• KSBB Journal
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• 제17권1호
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• pp.49-53
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• 2002

GABA가 고함유된 발아현미를 생산할 수 있는 전략을 마련하고자 현미발아시 통상적인 물발아구 외에 젖산발아구, glutamic acid발아구로 나누어 발아 이전의 현미와 GABA 및 일부 유리아미노산 함량을 비교분석하였다. 5 mM glutamic acid 용액을 발아에 사용한 경우 가장 높은 GABA 함량 증진을 보여 시료 g당 및 시료 추출물 중의 단백질 mg당 증가정도가 발아하지 않은 현미에 비해 각각 8배와 12배로 나타났다. 또한 glutamic acid 발아구는 물발아나 젖산발아시 현저히 감소되던 serine의 함량을 오히려 증진시켰다. 모든 발아구에서 GABA 및 alanine 함량이 증진된 것과 는 반대로 glutamic acid와 aspartic acid 함량은 현저히 감소 하였다. 이는 발아 과정에 의해 glutamic acid는 GABA로 aspartic acid는 alanine으로 전환된 것에 기인된 것이라 여겨진다. 이상의 결과를 종합하면 현미발아시 glutamic acid액을 사용하면 기능성 물질인 GABA 함량을 현저히 증진시키며, serine의 감소를 막을 수 있어 기능성이 보강된 발아현미를 얻을 수 있을 것으로 기대된다.

• BMB Reports
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• 제33권6호
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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.

• 대한화장품학회지
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• 제34권2호
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• pp.143-148
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• 2008

아미노산 자동분석기(amino acid auto analyzer, AAA)를 이용하여 모발의 탈색 및 퍼머넌트 웨이브 시술에 의한 모발시료의 17가지 아미노산(aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystein, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine) 함량변화를 조사하였다. 시료는 30대 남성 10명의 모발을 채취하였으며 $110^{\circ}C$ 24 h 동안 6 N 염산 10 mL로 가수분해 후 분석하였다. 실험결과 처치별 모발의 아미노산 함량은 정상모발 73.9%, 퍼머넌트 웨이브 모발 71.5% 및 탈색모발 69.3%로 분석되었고, 탈색효과에 의한 아미노산 함량 감소가 퍼머넌트 웨이브 효과에서 보다 크게 분석되었다. 특히 lysine과 tyrosine의 조성은 탈색이나 퍼머넌트 웨이브 모발에서 25% 가량 현저히 감소하였다. 반면에 serine, cystein, leucine 및 histidine의 함량변화는 일어나지 않았다.