• 미생물학회지
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• 제51권2호
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• pp.108-114
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• 2015

본 연구에서는 Streptomyces subrutilus P5의 생장과 세포내 외 철 함유 superoxide dismutase 활성을 비교 분석하여 철함유 superoxide dismutase의 분비 시점을 확인하고 분자 수준에서 이 효소의 분비에 관여하는 유전정보를 확인하고자 하였다. Streptomyces subrutilus P5의 균체 생장은 건체 중량을 측정하여 결정하였다. Glucose는 log phase에서 급격히 소모되어 24시간 후에 이르러 완전히 고갈되었다. 세포내의 철 함유 superoxide dismutase는 배양 후 3시간에 나타나며 세포외 철 함유 superoxide dismutase는 배양 후 7.5시간부터 나타난다. 따라서 superoxide dismutase는 용균에 의해서가 아니라 능동적인 분비기작에 의해서 세포 외로 분비된 것으로 추측할 수 있다. Streptomyces subrutilus P5의 sodF에는 signal peptide 유전정보가 존재하지 않았다. 그러나 sodF의 상류지역에서 다른 세균의 type III 분비단백질 유전자와 유사한 type III 분비상자가 발견되었다. Streptomyces 균주에서 type III 분비단백질이 존재할 가능성이 있음을 처음으로 제시하였다.

• Archives of Pharmacal Research
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• 제19권3호
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• pp.201-206
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• 1996

Nitric oxide (NO) is thought to be a second messenger involved in secretion. Upon stimulating pancreatic acinar cells with cholecystokinin-pancreozymin (CCK-PZ), NO formation has been shown to be associated with increased levels of cGMP (Seo et al., 1995). To elucidate the signaling pathway of VIP-induced enzyme secretion, we investigated the NO and cGMP synthesis steps as potential steps where two signal pathways triggered by CCK-PZ and VIP interact. The results obtained in this work provide evidence that increase in pancreatic enzyme secretion by treatment with VIP has no relationship with NOS activity and cGMP level. This conclusion was derived from the following findings that VIP treatment of rat pancreatic tissue increased amylase release as well as protein output in a dose- and time-dependent manner, whereas NOS activity and cGMP synthesis were not affected by VIP treatment as monitored by NOS activity assay and determining cGMP level, which was further confirmed by a NOS-inhibitor study. Consequently, CCK-PZ or VIP increases enzyme secretion in rat pancreatic tissue, but the two hormones are different in their mode of action. Together the results suggest that signaling pathway of VIP-induced enzyme secretion might either bypass the NO and cGMP synthesis steps or lie on a distinct pathway from CCK-PZ-induced pathway.

• 미생물학회지
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• 제29권5호
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• pp.267-269
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• 1991

The hyphal tip laccase of Coprinus congregatus which is a membrane-associated enzyme and shows diffdrdnt banding patterns of PAGE analysis when compared with the enzyme of liquid culture (Choi et al. 1987) has been successfully secreted to culture medium in liquid shake culture by lowering the pH of medium to 4.0. When the fungus is cultivated in YpSs(pH 4.0) liquid, only the hyphal tip laccase is found in the medium after 6 hr incubation and there is no liquid-type enzyme when examined by PAGE analysis.

• 한국가금학회지
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• 제16권4호
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• pp.219-232
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• 1989

본 연구는 닭에 있어서 사료성분에 대한 췌장소화효소(amylase. trypsinogen 및 chymotrypsinogen) 분비기구에 대해서 검토했다. 먼저, 췌효소분비의 단기응답실험에 유용한 새로운 췌액채취법을 개발했다. 이 방법을 이용해서 아미노산 및 glucose를 날개정맥으로 투여한 결과 phenylalanine만이 trypsinogen 및 Chymotrypsinogen이 증가되었지만 그 외의 아미노산 및 glucose에 의해서는 분비증가 효과가 없었다. Cholecystokinin(CCK)투여 에 의해 췌효소분필는 즉각적으로 높은 분비반응을 보였으며, 이 반응은 또한 농도의존성을 나타냈다. CCK투여는 chymotrypsinogen의 쪽이 amylase 및 trypsinogen보다 높은 비율로 분비되는 선택적인 분비반응을 나타냈다. 아미노산과 CCK을 공동투여하면 첨가한 아미노산의 종류에 따라 췌효소분비반응은 여러 가지 형태로 증가되었지만 glucose와의 공동투여에서는 CCK 단독투여와 비교해서 차가 없었다. Valine과 arginine을 여러 가지 농도로 CCK와 공동 투여한 결과, valnine에서는 0.5mM일때, arginin에서는 5mM일때 가장 높은 분비반응을 보였다. 위의 결과로부터 아미노산의 조합에 의한 췌효소분비반응에 대해서 검토했다. 즉, 아미노산 mixture, threonine＋phenylalanine＋isoleucine, Threonine＋phenylalanine, threonine＋isoleucine 및 phenylalanine＋isoleucine과 CCK를 공동투여 했다. 각 물질을 투여한 후 50분간 분비한 효소를 비교하면, threonine＋phenylalanine에 의한 췌효소분비반응은 아미노산 mixture에 의한 분비반응과 동일하게 높은 반응을 보였다. 이상의 결과로부터 닭에 있어서 췌장소화효소분비는 CCK와 아미노산의 사이에 협동작용이 있으며, 그 협동작용은 아미노산의 종류에 따라 선택적인 분비반응을 함으로써 장내소화가 진행된다고 본다.

• 대한약리학회지
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• 제13권2호
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• pp.41-48
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• 1977

In 1968, Case et al. first studied the importance of cyclic AMP as an intermediate in the action of secretin and cholecystokinin-pancreozymin and they suggested that the action of secretin, not that of cholecystokinin-pancreozymin, may be mediated through cyclic AMP. Recently Albano et al. reported that in the exocrine pancreas each of the two major physiological functions is modulated a specific cyclic nucleotide, enzyme secretion by cyclic GMP, and fluid and ionic secretion by cyclic AMP. But in pancreas still conflicting results have been reported on the role of cyclic nucleotides in enzyme and electrolyte secretion. In these study, the role of cyclic nucleotides in the exocrine pancreatic secretion was examined. The results are as follows. 1) Very strong stimulation on amylase release from guinea pig pancreatic slice was produced by 1 unit of cholecystokinin-pancreozymin but as compared to that of cholecystokinin-pancreozymin very weak response was observed by 1 unit of secretion or $1\;{\mu}g$ of VIP. 2) Both cholecystokinin-pancreozymin and acetylcholine produced a rapid and marked rise in cyclic GMP as well as cyclic AMP in isolated pancreatic tissue. However, both secretin and VIP failed to alter significantly the basal level of cyclic GMP in pancreatic fragments. 3) Atropine inhibited acetylcholine mediated amylase release, but did not affect the cholecystokinin-pancreozymin response. Furthermore, atropine pretreatment produced a marked inhibitory effect on the increase of tissue cyclic nucleotides induced by cholecystokinin-pancreozymin and acetylcholine. In summary, these results suggest that whereas the pancreatic secretion produced by secretin and VIP is modulated by the formation of cyclic AMP, the pancreatic enzyme secretion in response to cholecystokinin-pancreozymin and acetylcholine is triggered by both cyclic AMP and cyclic GMP.

• 대한한방내과학회지
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• 제31권1호
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• pp.25-39
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• 2010

Background : At high glucose levels in $\beta$-cells, cell viability and insulin secretion are decreased by glucotoxicity. Sopyung-tang(SPT) had an effect on blood glucose level decrease and antioxidant enzyme activities in streptozotocin-induced diabetic rats. Objectives : This study performed a series of experiment to verify the effects of SPT extract on the cell viability, antioxidant enzyme activities, insulin secretion and insulin mRNA expression at hyperglycemic states of RIN-m5F. Methods : After treatment at various concentrations of SPT added to the RIN-m5F cells, cell viability by MTT assay, free radical-scavenging activity, SOD activity and insulin secretion were measured. Additionally, insulin-related gene expression was measured using real-time RT-PCR. Results : Compared to the control group, SPT extract showed considerable effects on RIN-m5F cell viability, DPPH radical-scavenging activity, superoxide dismutase (SOD) activity, insulin secretion and insulin-related gene expression. Conclusions : This study showed that SPT extract has an effect on $\beta$-cell cell viability, insulin secretion and insulin-related gene expression. Thus, SPT extract may be used for treatment of diabetes and its complications. Further mechanism studies of SPT seem to be necessary on the glucotoxicity and oxidative stress.

• 대한약리학회지
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• 제25권1호
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• pp.75-85
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• 1989

면역 보체가 결합되어 있는 zymosan에 의하여 활성화된 다형핵 백혈구에서 세포 투과성 물질인 N-ethylmaleiamide과 $Hg^{++}$은 superoxide 라디칼 생성, NADPH oxidase 활성도 및 lysosomal enzyme (lactic dehydrogenase, ${\beta}-glucuronidase$)의 유리를 억제하였다. 세포막 단백에 특이적인 p-chloromercuribenzoic acid와 p-chloromercuribenzenesulfonic acid는 superoxide 라디칼 생성에 영향을 주지 않았으나 NADPH oxidase 활성도와 lysosomal enzyme의 유리를 억제하였다. 식작용 중에 세포막과 세포내의 sulfhydryl기는 반응시간에 따라 점진적으로 감소하였다. N-ethylmaleiamide와 $Hg^{++}$은 세포막과 세포내의 sulfhydryl기를 모두 감소시켰다. P-Chloromercuribenzoic acid와 p-chloromercuribenzenesulfonic acid는 세포막의 sulfhydryl기를 유의하게 감소시켰으나 세포내 용해성 sulfhydryl기에는 영향을 주지않았다. Cysteine과 mercaptopropionylglycine는 superoxide 라디칼의 생성과 lysosomal enzyme의 유리를 억제하였다. Gluthathione은 superoxide생성에 영향을 주지 않았으나 뚜렷하게 lactic dehydrogenase의 유리를 억제하였다. N-ethylmaleiamide에 의한 superoxide 생성의 억제는 cysteine과 mercaptopropionyl-glycine에 의하여 반전되었으나 gluthathione의 영향은 없었다. N-ethylamleiamide에 의한 NADPH oxidase의 비활성화는 gluthathione, cysteine과 mercaptopropionylglycine에 의하여 저해되었다. Carbachol에 의하여 항진된 superoxide 라디칼 생성은 N-ethylamleiamide에 의하여 완전히 억제되었고, atropine에 의하여 길항되었다. 그러므로, 외부 자극에 대한 다형핵 백혈구 반응의 표현은 sulfhydryl기의 양의 변화와 연관이 있을 것으로 시사되었다. Lysosomal enzyme 유리는 세포막과 세포내의 sulfhydryl기에 의하여, 이에 반하여 superoxide생성은 세포내 sulfhydryl기에 의해서 영향받을 것으로 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제21권4호
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• pp.412-420
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• 2011

Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 ${\mu}g$/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at $50^{\circ}C$, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.

• Tuberculosis and Respiratory Diseases
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• 제56권3호
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• pp.289-296
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• 2004

연구배경 : 기도 질환에서 점액이 과량 분비되는 경우 환자에게 불편함을 줄뿐만 아니라 기도 질환 예후에도 나쁜 영향을 미친다. 그러나, 기도 질환에서 점액이 과량 분비되는 것을 효과적으로 막는 방법이 없다. 점액의 성분 중 mucin은 당화 단백질로서 점액이 점성을 띄게 만드는 주요 성분이다. 본 연구를 통해서 mucin 분비 기전에 proteinase가 관여하는지 확인하고 만일 proteinase가 mucin 분비기전에 관여 한다면 어느 proteinase가 그런 역할을 하는지 확인하고자 하였다. 방 법 : (1) mucin 분비 억제 실험 군 특이적 proteinase 억제제를 사용하여 어느 군에 속하는 proteinase가 mucin 분비를 억제하는지 mucin을 생산하는 폐 세포주인 Calu-3를 이용하여 알아보았다. 군 특이적 proteinase 억제제로 PMSF(phenylmethylsulfonyl fluoride, serine proteinase inhibitor), E-64(cysteine proteinase inhibitor), Pepstatin(aspartic proteinase inhibitor), 1,10-Phenanthroline(metalloproteinase inhibitor)를사용하였다. 군 특이적 억제제를 Calu-3에 24시간동안 처리하여 분비된 mucin양을 enzyme linked immunoabsorbant assay(MUC5AC)로 정량하였고 그 결과를 대조군과 비교하였다. (2) Mucin 분비 자극 실험 Metalloproteinase 중에서 기도 질환 발병과 관련 있다고 알려진 matrix metalloproteinase-9 (MMP-9), MMP-12 그리고 TNF-alpha converting enzyme(TACE)를 Calu-3에 24시간 처리하여 분비된 mucin양을 enzyme linked immunoabsorbant assay (MUC5AC)로 정량하였고 그 결과를 대조군과 비교하였다. 결 과 : (1) 군 특이적 proteinase 억제제인 PMSF($10^{-4}M$), E-64($10^{-4}M$), Pepstatin($10^{-6}M$), 1,10-Phenanthroline($10^{-4}M$)는 MUC5AC 분비를 각각 $1{\pm}4.9%$(평균${\pm}$표준오차; 대조군과 비교 시 P=1.0), $-6{\pm}3.9%$ (P=0.34), $-13{\pm}9.7%$(P=0.34), $41{\pm}8.2%$(P=0.03) 감소시켰다(실험 회수 4번). (2) MMP-9(250ng/ml), MMP-12(100ng/ml), TACE(200ng/ml)에 의한 MUC5AC 분비량은 대조군에 비하여 각각 $103{\pm}6%$(P=0.39), $102{\pm}8%$(P=1.0), $107{\pm}13%$(P=0.39)이었다(실험 회수 6번). 결 론 : mucin 분비 기전에 metalloproteinase가 관여함을 시사하지만 MMP-9, MMP-12, TACE는 in vitro 모델에서 mucin 분비에 영향을 미치지 않았다.

• BMB Reports
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• 제44권1호
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• pp.40-45
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• 2011

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects cells and tissues from extracellular damage by eliminating superoxide anion radicals produced during metabolism. Two different forms of EC-SOD exist, and their different enzyme activities are a result of different disulfide bond patterns. Although only two folding variants have been discovered so far, five folding variants are theoretically possible. Therefore, we constructed five different mutant EC-SOD expression vectors by substituting cysteine residues with serine residues and evaluated their expression levels and enzyme activities. The mutant EC-SODs were expressed at lower levels than that of wild-type EC-SOD, and all of the mutants exhibited inhibited extracellular secretion, except for C195S ECSOD. Finally, we demonstrated that co-expression of wild-type EC-SOD and any one of the mutant EC-SODs resulted in reduced secretion of wild-type EC-SOD. We speculate that mutant EC-SOD causes malfunctions in systems such as antioxidant systems and sensitizes tissues to ROS-mediated diseases.