• 미생물학회지
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• 제38권4호
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• pp.218-218
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• 2002

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• Journal of Microbiology
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• 제38권4호
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• pp.218-223
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• 2000

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.

• 한국식품과학회지
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• 제21권1호
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• pp.86-91
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• 1989

Streptococci의 배양중의 염의 농도나 배지내 질소원의 조성 등이 쓴 맛 펩타이드의 형성에 깊이 관여한다고 알려진 세포내 및 세포외 프로테이나제(ICP, ECP)의 역가에 미치는 영향을 조사하기 위해 Streptococcus lactis $ML_8$ 및 Streptococcus cremoris $ML_4$ 두 균주를 NaCl 농도 $0{\sim}4%$, 배지 질소원중 카제인 함량을 $0{\sim}100%$로 달리한 조건에서 배양하고 그 때의 생육도와 균체내 및 균체외 프로테이나제의 역가를 측정하고 또한 같은 균체수에서의 프로테이나제 역가를 비교하기 위해 $10^{10}$ 세포당 효소의 역가도 계산하였다. 배지중 NaCl농도가 증가함에 따라 균체의 생육과 ECP의 역가는 감소했으나 ICP의 경우 $10^{10}$ 세포당 역가는 오히려 증가하였다. 이는 배지중 NaCl이 세포벽과 세포막에 관련된 ECP의 역가에만 직접 영향을 미치고 ICP의 역가에는 큰 영향을 미치지 않음을 시사하는 것으로 생각되었다. 배지의 질소원중 미분해 단백질인 카제인의 함량을 높게 하였을 때 균체의 생육은 낮았으나 효소 생산계 촉진에 의해 ECP의 역가는 두 균주 모두 높아졌다.

• BMB Reports
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• 제40권5호
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• pp.678-683
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• 2007

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 ${\mu}g/ml$)-stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metallo-proteinase-9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular $Ca^{2+}$ mobilization, and thromboxane $A_2$ ($TXA_2$) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or $TXA_2$ synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular $Ca^{2+}$ mobilization and $TXA_2$ production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.

• Journal of Microbiology
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• 제35권3호
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• pp.213-221
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• 1997

The secretion of the alkaliphilic Bacillus sp. S-1 extracellular pullulanase involves translocation across the cytoplasmic membrane of the Gram-positive bacterial cell envelope. Translocation of the intracellular pullulanase PUL-I, was traced to elucidate the mechanism and pathway of protein secretion from an alkaliphilic Bacillus sp. S-1. Pullulanase could be slowly bue quantitatively released into the medium during growth of the cells in medium contianing proteinase K. The released pullulanase lacked the N-terminal domain. The N-terminus is the sole membrane anchor in the pullulanase protein and was not affected by proteases, confirming that it is not exposed on the cell surface. Processing of a 180,000M$\_$r/ pullulanase to a 140,000M$\_$r/ polypeptide has been demonstrated in cell extracts using antibodies raised against 140,000M$\_$r/ extracellular form. Processing of the 180,000 M$\_$r/ protein occured during the preparation of extracts in an alkaline pH condition. A modified rapid extraction procedure suggested that the processing event also occured in vivo. Processing apparently increased the activity of pullulanase. The western blotting analysis with mouse anti-serum against 140-kDa extracellular pullulanase PUL-E showed that PUL-I is processed into PUL-X via intermediate form of PUL-E. Possible explanationa for the translocation are discussed.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.59-67
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• 2023

Thrombin is a serine protease that participates in a variety of biological signaling through protease-activated receptors. Intestinal myofibroblasts play central roles in maintaining intestinal homeostasis. In this study, we found that thrombin-induced apoptosis is mediated by the calcium-mediated activation of cytosolic phospholipase A₂ in the CCD-18Co cell. Thrombin reduced cell viability by inducing apoptosis and proteinase-activated receptor-1 antagonist attenuated thrombin-induced cell death. Endogenous ceramide did not affect the cell viability itself, but a ceramide-mediated pathway was involved in thrombin-induced cell death. Thrombin increased intracellular calcium levels and cytosolic phospholipase A₂ activity. The ceramide synthase inhibitor Fumonisin B1, intracellular calcium chelator BAPTA-AM, and cytosolic phospholipase A₂ inhibitor AACOCF₃ inhibited thrombin-induced cell death. Thrombin stimulated arachidonic acid release and reactive oxygen species generation, which was blocked by AACOCF₃, BAPTA-AM, and the antioxidant reagent Trolox. Taken together, thrombin triggered apoptosis through calcium-mediated activation of cytosolic phospholipase A₂ in intestinal myofibroblasts.

• 한국식품과학회지
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• 제34권2호
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• pp.311-318
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• 2002

Lactobacillus plantarum에 대해 항균활성을 지니는 유산균을 김치로부터 분리하였다. 이 균은 Leuconostoc mesenteroides으로 동정되었으며 Leuconostoc mesenteroides B7으로 명명하였다. Leuconostoc mesenteroides B7이 생산하는 박테리오신은(박테리오신 B7) pH 및 열 안정성이 뛰어나 pH $2.5{\sim}9.5$ 그리고 $4^{\circ}C{\sim}120^{\circ}C$ 열처리에서도 항균활성을 안정하게 유지하였다. 박테리오신 B7은 proteinase K, trypsin, ${\alpha}-chymotrypsin$, 그리고 protease 처리에 의해 항균활성이 실활되므로 peptide나 단백질로 이루진 구조임을 알 수 있었다. 정제된 박테리오신 B7은 Tricine-SDS-PAGE를 통하여 분자량이 약 3,500 dalton임을 확인하였다. Leuconostoc mesenteroides B7과 이에 감수성인 균주, Lb. plantarum KFRI 464 혹은 Lb. delbruekii KFRI 347와의 혼합배양에 의하여 박테리오신 B7 생산이 증가됨을 확인하였고, 이는 박테리오신 생산 유도물질이(inducing factor) 감수성균주내에 존재함을 시사한다. 감수성균주의 세포분획을 통하여 inducing factor는 감수성균주의 세포벽과 세포내에 존재함을 알았다. 이 inducing factor가 proteinase K처리로 박테리오신 유도활성을 상실함으로써 이는 단백질성물질임을 시사하였다.

• 한국미생물·생명공학회지
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• 제34권3호
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• pp.221-227
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• 2006

박테리오신 BSCX1은 Bacillus subtilis cx1에 의해 생산되는 항균성 peptide이다. B. subtilis cx1의 박테리오신(BSCX1)은 Bacilius subtilis ATCC6633, Listeria monocytogenes KCTC3569를 포함한 그람양성균과 Salmonella typhi ATCC19430, Escherichia coli ATCC25922와 같은 그람음성균에 대해서도 비교적 넓은 항균활성 범위를 가진다. 박테리오신 생산균주인 B. subtilis cx1과 그것의 감수성 균주인 B. subtilis ATCC6633을 공동 배양한 결과, 박테리오신 BSCX1의 생산이 증가됨을 확인할 수 있다. 이 결과는 박테리오신 생산균주 B. subtilis cx1의 성장 배지내에 박테리오신 감수성 균주가 존재함이 BSCX1 생산을 촉진시키는 것을 의미한다. 감수성 균주의 박테리오신 유도 작용을 확인하였으므로 유도물질이 감수성 균주의 어느 위치에 존재하는지 밝히기 위해 B. subtilis ATCC6633을 분획하여 실험한 결과 세포내 분획과 세포파쇄물에 모두 유도물질이 존재함을 확인하였다. BSCX1 유도물질의 유도활성은 pH 2.5에서 pH 9.5에 걸쳐 전 구간에서 유지되었으며, $50^{\circ}C$이상에서는 3시간 이내에 불활성화 되었다. 유도물질에 단백분해효소인 proteinase K를 처리한 결과 유도활성이 사라져 단백질성 물질임을 알 수 있었다.

• 환경생물
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• 제22권4호
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• pp.487-493
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• 2004

To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA sequencing. It has an antifungal activity against 9 plant pathogenic fungi in a paper disc assay. The antifungal activity- deficient mutant, B. subtilis mLTD was induced at a 5 kGy dose of $^{60}Co$ gamma radiation. Using the two-dimensional electrophoresis and the matrix assisted laser desorption ionization time-of-flight mass spectrometry, the comparison analysis of proteins between the wild and mutant were performed. A major intracellular serine proteinase IspA (MW: 32.5 kDa), a NAD (P) H dehydrogenase (MW: 20.0 kDa), and a stage II sporulation protein AA, SpoIIAA (MW: 14.3kDa) were detected only in the B. subtilis LTD. These results suggested that the functions of these proteins found only in the B. subtilis LTD could. be closely related to the antifungal activity against plant pathogenic fungi.