• Journal of Pharmaceutical Investigation
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• 제24권1호
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• pp.1-9
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• 1994

In order to study systemic peptide delivery through the ocular route, the stabilities of methionine enkephalin (Met-Enk) and $[D-ala^2]-methionine$ enkephalinamide (YAGFM) in the corneal extracts of rabbits were investigated using reversed phase HPLC. Met-Enk was found to be hydrolyzed most rapidly in the corneal epithelium, but YAGFM was relatively stable. Aminopeptidases appeared to contribute over 60% to the degradation of Met-Enk and the degradation rate of Met-Enk followed the first order kinetics. The half-lives of Met-Enk in the extracts of the corneal epithelium and endothelium were 36 and 673 min, respectively. From the effects of enzyme inhibitors, it was found that the application of the mixture of amastatin, thimerosal and EDTA was very useful for the inhibition of peptide degradation.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.530-533
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• 2002

This paper describes the development of an enzymatic assay system for the identification of specific inhibitors of sortase, a transpeptidase that cleaves surface proteins of Cram-positive bacteria, from Staphylococcus aureus ATCC 6538p for antibacterial drug discovery. The coding region of the enzyme was amplified with the exception of the N-terminal membrane anchor sequence, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The enzyme activity was determined by quantifying increased fluorescence intensity upon cleavage of synthetic Dabcyl-QALPETGEE-Edans peptide. The results suggest that the developed in vitro assay system call be used in the search for sortase inhibitors In a short period of time.

• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.117-120
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• 2014

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.

• The Korean Journal of Physiology and Pharmacology
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• 제20권5호
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• pp.515-523
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• 2016

Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The ${\beta}1$-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1047-1052
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• 2006

A bile salt hydrolase (BSH) was purified from Lactobacillus plantarum CK 102 and its enzymatic properties were characterized. This enzyme was successfully purified using ion-exchange chromatography with Q-Excellose and hydrophobic interaction chromatography with Butyl-Excellose. The purified enzyme showed a single protein band of 37 kDa by SDS-polyacrylamide gel electrophoresis, which was similar to the molecular weight of known BSHs. The amino acid sequence of GLGLPGDLSSMSR, determined by MALDI-TOF, was identical to that of BSH of L. plantarum WCFS1. Although this BSH hydrolyzed all of the six major human bile salts, glycine-conjugated bile acid was the best substrate, based on its specificity and $K_{m}$ value. Among the various substrates, the purified enzyme maximally hydrolyzed glycocholate with apparent $K_{m}$ and $V_{max}$ values of 0.5 mM and 94 nmol/min/mg, respectively. The optimal pH of the enzyme ranged from 5.8 to 6.3. This enzyme was strongly inhibited by thiol enzyme inhibitors such as iodoacetate and periodic acid.

• 한국동물학회지
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• 제34권2호
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• pp.142-147
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• 1991

드렁허리의 혈장에서 얻은 angiotensin I-converting enzyme(ACE)의 활성도를 포유동물과 비교하였다. 드렁허리의 혈장내 ACE는 pH 10에서 가장 높은 활성도를 나타냈으며 포유동물에서 보다 알칼리성이었다. 또한 최적활성도를 나타내는 데 필요한 C1 이온의 요구성은 종에 따라 다르게 나타났다. 드렁허리의 ACE 활성도는 ACE의 활성억제제인 EDTA, teprotide 및 captopril에 의해서 이들의 농도에 따라 억제 되었으며, 이 억제현상은 포유동물의 혈장 ACE와 매우 비슷하였다. 드렁허리의 ACE 활성도는 cobalt에 의하여 증가되었으며, 포유동물에 비해서 열에 불안정하였다. 또한 여러 조직중에서느 뇌에서 가장 높은 ACE 활성도가 측정되었다.

• Journal of Preventive Medicine and Public Health
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• 제7권1호
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• pp.107-113
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• 1974

위생공학에서 하수나 공장폐수를 처리할 때 이용하는 생물학적 처리과정은 미생물에서 생성되는 효소에 의하여 이루어 진다. 본 연구에서는 diastase의 활성에 대하여 수소이온 농도, 온도, 기질 농도 및 방해물질 등 여러가지 요인이 미치는 영향을 실험적으로 관찰하여 다음과 같은 결론을 얻었다. (1) diastase의 활성도는 pH $4.0{\sim}7.0$의 범위가 적당하였고 최적치는 pH 5.0이었다. (2) diastase의 활성도는 $30^{\circ}C{\sim}50^{\circ}C$ 높았으며 최적온도는 $40^{\circ}C$였다. (3) 기질농도의 영향을 보면 녹말농도 $750{\mu}g/ml$까지는 diastase의 활성도가 급격히 증가하였고, 그 이후에는 한정속도를 나타냈다. Michaelis 방정식을 적용시켜 보면 한정속도는 $415{\mu}g/ml$ starch removed/ml of reaction mixture/min였고 Michaelis 상수는 $340{\mu}g/ml$였다. (4) 방해물질의 영향을 보면 NaCl 농도 1.0%, $HgCl_2$ 농도 0.001%에서 diastase의 활성은 완전히 억제되었다.

• 생명과학회지
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• 제34권9호
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• pp.656-665
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• 2024

효소 억제제는 인체 내 다양한 대사과정을 조절하는 단백질에 속하는 효소의 작용을 억제하여 특정 질병이나 상태를 치료하는 데 도움을 준다. 퀘르세틴은 폴리페놀 계열에 속하는 플라보노이드의 일종으로 식물에서 발견되는 이차 대사산물이다. 이러한 퀘르세틴은 구조적 이점을 바탕으로 효소의 활성부위에 결합하여 α-glucosidase, acetylcholinesterase, bacterial neuraminidase, xanthine oxidase에 대한 억제 활성을 갖는 것으로 보고된다. 또한, 퀘르세틴은 glycosylation, methoxylation, alkylation 등을 통해 특징적인 치환기를 가질 수 있으며 이러한 천연 퀘르세틴 유도체는 효소 활성 부위의 촉매 잔기와 특이적인 결합 패턴을 나타내어 더욱 우수한 효소저해활성을 가질 수 있다. 따라서, 본 논문에서는 퀘르세틴 및 그 유도체의 특징적인 구조에 대해 알아보고 이들의 효소저해활성을 통해 유도체별 다양한 질병을 목표로 하는 효과적인 효소 저해제 개발에 유망한 후보가 될 수 있음을 시사한다.

• Bulletin of the Korean Chemical Society
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• 제26권2호
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• pp.285-291
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• 2005

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is the viral RNA-dependent RNA polymerase (RdRp), which is the essential catalytic enzyme for the viral replication and is an appealing target for the development of new therapeutic agents against HCV infection. A small amount of serum from a single patient with hepatitis C was used to get the genome of a Korean HCV isolate. Sequence analysis of NS5B 1701 nucleotides showed the genotype of a Korean isolate to be subtype 1b. The soluble recombinant HCV NS5B polymerase lacking the C-terminal 24 amino acids was expressed and purified to homogeneity. With the highly purified NS5B protein, we established in vitro systems for RdRp activity to identify potential polymerase inhibitors. The rhodanine family compounds were found to be potent and specific inhibitors of NS5B from high throughput screening (HTS) assay utilizing the scintillation proximity assay (SPA) system. The binding mode of an inhibitor was analyzed by measuring various kinetic parameters. Lineweaver-Burk plots of the inhibitor suggested it binds not to the active site of NS5B polymerase, but to an allosteric site of the enzyme. The activity of NS5B in in vitro polymerase reactions with homopolymeric RNA requires interaction with multiple substrates that include a template/primer and ribonucleotide triphosphate. Steady-state kinetic parameter, such as Km, was determined for the ribonucleotide triphosphate. One of compounds found interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitively with respect to UTP. Furthermore, we also investigated the ability of the compound to inhibit NS5B-directed viral RNA replication using the Huh7 cell-based HCV replicon system. The investigation is potentially very useful for the utility of such compounds as anti-hepatitic agents.

• Archives of Pharmacal Research
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• 제18권5호
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• pp.325-331
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• 1995

The objective of this study was to find a rational dosage form for vaginal mucosal delivery of LHRH. Vaginal absorption of LHRH was estimated by measuring its ovulation inducting effect in rat and in vitro vaginal membrane permeation study in rabbit. THe effects of different hydrogel bases, such as Polycarbophil and Pemulen compared with solutions on vaginal membrane permeation of LHRH were investigated. Sodium laurate, disodium ethylenediamine brane permeaiton of LHRH were investigated. Sodium ethylenediamine tetraacetate (EDTA) and sodium tauro-24, 25-dihydrofusidate (STDHF), which are effective peptidase inhibitors were chosen as additives to a LHRH hydrogel delivery system and LHRH solutions. A Polycarbophil compared with a solution formulation 3.4 times increase in LHRH vaginal membrane permeability compared with a solution formulation. Vaginal membrane permeability from the Polycarbophil was greater than that from Pemulen hydrogels. This may be due to the larger bioadhesive values. LHRH solution with EDTA(2%), STDHF(1%) and sodlaurate(0.5%) showed 4.1 times, 4.8 times and 6.0 times of ovulation inducing activity compared with control. These results suggest that enzyme inhibition effect of EDTA, STDHF and sod, laurate may be result in substantial enhancement of vaginal absorption. By administraiton of Polycarbophil hydrogels containing LHRH the ovulation inducing activity was 3.3 times greater than the solutions. This result indicates the bioadhesive hydrogels as well as peptidase in hibition significantly improved absorption of LHRH. By coadministration with these inhibitors the ovulation inducing activity of Polycarbophi hydrogel containing LHRH was comparable with subcutaneous administration in ovulation inducing activity.