• Journal of Pharmaceutical Investigation
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• v.24 no.1
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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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• v.12 no.3
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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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• v.52 no.1
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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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• v.20 no.5
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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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• v.16 no.7
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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.

• The Korean Journal of Zoology
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• v.34 no.2
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• pp.142-147
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• 1991

Angiotensin I-converting enzyme (ACE) activity has been characterized in the rice eel, Monopterus albus. Peak activity of ACE in plasma from the rice eel was shown at around pH 10, which was more alkaline compared to that of mammals. Chloride requirements for the optimal ACE activity were different from species to spedes. ACE inhibitors, EDTA, teprotide (SQ 20, 881), and captopril (SQ 14, 225) showed dose-dependent inhibitions of ACE activity in plasma from the rice eel as well as mammals. ACE activity in the rice eel was increased by CoCI2, and the enzyme activity was more unstable at high temperature as compared to mammals The highest activity of ACE among the various tissues in the rice eel was found in the brain.

• Journal of Preventive Medicine and Public Health
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• v.7 no.1
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• pp.107-113
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• 1974

Many factors exert an influence on enzyme activity and thus on the rate of reactions that they catalyse. The most important of these factors are pH, temperature, substrate concentration, and the concentration of some inhibitors present. A solution of the enzyme diastase, which breaks down molecules of the polysaccharide starch to the disaccharide maltose by hydrolysis, was provided. Activity of this enzyme was measured by the rate at which starch was removed from the reaction mixture. These experiments were designed to study this reaction rate under varying conditions and the following results were obtained. 1. The range of optimum pH for this enzyme at room temperature was 4.0-7.0 and the optimum pH was 5.0. 2. The range of optimum temperatures for this enzyme at pH 7.0 was $30^{\circ}C-50^{\circ}C$ and the optimum temperature was $40^{\circ}C$. 3. The relationship between the enzyme activity and substrate concentration could be expressed by the Michaelis-Menten equation. The limiting velocity of this enzyme at room temperature and pH 7.0 was $415{\mu}g$ starch removed/ml of reaction mixture/min and $K_m$, Michaelis constant, was $343{\mu}g/ml$. 4. Inhibitors NaCl and $HgCl_2$ blocked this enzyme activity completely at 1% and 0.01% respectively.

• Bulletin of the Korean Chemical Society
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• v.26 no.2
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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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• v.18 no.5
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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.

• BMB Reports
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• v.33 no.4
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• pp.321-325
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• 2000

Quinone reductase was purified to homogeneity from bovine liver by using ammonium sulfate fractionation, ionexchange chromatography, and gel filtration chromatography. The enzyme utilized either NADH or NADPH as the electron donor. The enzyme catalyzed the reduction of several quinones and other artificial electron acceptors. Furthermore, the enzyme catalyzed NAD(P)H-dependent reduction of azobenzene. The apparent Km for 1,4-benzoquinone and azobenzene was 1.64 mM and 0.524 mM, respectively. The reduction of azobenzene by quinone reductase was almost entirely inhibited by dicumarol or Cibacron blue 3GA, potent inhibitors of the mammalian quinone reductase. In the presence of 1.0${\mu}M$ Cibacron blue 3GA, azoreductase activity was lowered by 45%, and almost complete inhibition was seen above 2.0 ${\mu}M$ Cibacron blue 3GA.