• Bulletin of the Korean Chemical Society
• /
• v.26 no.3
• /
• pp.433-439
• /
• 2005

To gain further insight on the relationship between structure and functions of glutathione S-transferase (GST), the three tyrosine 108 mutants, Y108A, Y108F, and Y108W, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity, but approximately 63% increase of steroid isomerase activity toward ${\Delta}^5$–[androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of $k_{cat}\;and\;k_{cat}/K_m{^{EPNP}}$ by 2 orders of magnitude, suggesting that Tyr 108 residue of hGSTP1-1 are considered to be important for the catalysis and the binding of the epoxide substrates. The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP, cumene hydroperoxide and ${\Delta}^5$–ndrostene 3,17-dione, but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB. We conclude from these results that Tyr 108 in hGST P1-1 plays very different roles depending upon the nature of the electrophilic substrates.

• Korean Journal of Pharmacognosy
• /
• v.22 no.2
• /
• pp.101-111
• /
• 1991

Polyphenol oxidase(PPO) was purified from an extract of Puerariae Radix by ammonium sulfate fractionation followed by Sephadex G-150 column chromatography, which resulted in a 56-fold increase in specific activity. The enzyme was optimum of pH 6.5. The optimum temperature of enzymic reaction was about $40^{\circ}$. The enzyme was thermostable with a half-life equal to 32 min at $70^{\circ}$. Km values of the PPO for catechol and pyrogallol from Lineweaver Burk plots were $1.3{\times}10^{-2}M$, $1.16{\times}10^{-2}M$, respectively. The substrate specificity of the Puerariae Radix PPO showed high affinity toward pyrogallol. Reducing reagents such as cysteine, potassium metabisulfite, ascorbic acid, 2-mercaptoethanol completely inhibited the PPO activity at $10^{-2}M$ level. Linewear-Burk analysis of inhibition data revealed that the inhibition by cysteine, 2-mercaptoethanol, 4-nitrocatechol, potassium cyanide was competitive with Ki values of $4.3{\times10^{-2}M,\;0.73{\times}10^{-6}M,\;6.9{\times}10^{-6}M,\;6.4{\times}10^{-7}M$, respectively. The browning reaction by PPO was observed to decrease temporarily with the addition of sodium diethyl dithiocarbamate, a well known copper chelating agent. Among the divalent cations, $Cu^{2+}$ ion was strong activator on PPO and $Mn^{2+},\;Co^{2+}$ ions was effect on PPO activity. $Zn^{2+},\;Mg^{2+}$ ions was inhibitor on PPO.

• Korean Journal of Veterinary Research
• /
• v.29 no.4
• /
• pp.503-515
• /
• 1989

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to avian infections bronchitis virus(IBV) were standardized. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV Mass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well($100{\mu}l$) and the plates were coated by completey drying at $37^{\circ}C$. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in $100{\mu}l$ volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive(IRP) serum. After repeated titration of IRP and negative serum, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive(S/P) OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; $Log_{10}$ ELISA titer=$5.568({\log}_{10}S/P)+4.161$ Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's.

• Korean Journal of Veterinary Research
• /
• v.39 no.5
• /
• pp.951-960
• /
• 1999

We performed this study to evaluate the potential clinical marker of urinary trypsinogen-2 together with amylase, lipase and urinary amylase creatinine clearance ratio (ACCR) for the diagnosis of acute pancreatitis in dogs. In the experiment on daily changing patterns of amylase, lipase and ACCR measurements in experimentally induced pancreatitis dogs, compared to values measured in pre-induction state, significant difference was seen in amylase until 5th day of induction, and for lipase significant difference was found during the 7th day of observation period (p < 0.05). No significant difference was found in ACCR for the study period (p > 0.05). On SDS-PAGE analysis of urine from experimentally induced pancreatitis dog, The 26kd band was markedly increased compared with that of normal state and that band was confirmed trypsinogen-2 using substrate interaction and isoelectric focusing assay after being eluted. When assessing the appearance of 26kd band on urine SDS-PAGE 87.1% (range: 50~100%) of experimentally induced pancreatitis dogs showed positive results, whereas no corresponding band was seen in dog without pancreatic disorders. With this result, determination of urinary trypsinogen-2 assay was found to have a high diagnostic value with a 70% of sensitivity and 100% of specificity as a routine test for pancreatitis, although the detection of trypsinogen-2 in urine can be varied on the progression stage of pancreatitis at the initial visit to animal clinic. We therefore suggest that the promising results in this study be used for the development of dipstick test for detecting acute pancreatitis in the future research.

• Proceedings of the Korean Vacuum Society Conference
• /
• 1999.07a
• /
• pp.203-203
• /
• 1999

Mass spectrometry technique provides the molecular weight distribution, data on the sequence of repeat units, polymer additives, and impurities, and structural information. time-of-Flight secondary Ion Mass Spectrometry (TOF-SIMS) has been used for structural characterization of various polymers1-2. the masses of repeat units and terminal groups and molecular weight distributions of polymers have been determined from their TOF-SIMS spectra. TOF-SMIS provides good sensitivity and structural specificity for high mass ions so that intact oligomers and large polymer fragments are observed. In this study, we investigated the detailed structural information on the oligomers and fragment ions of branched poly(1,3-butylene adipate) and branched poly[di(ethylene glycol) adipate] and the transesterification products of branched polyesters with trifluoroacetic acid or chloro difluoroacetic acid. Branched polyesters were chosen because they are important polymers but difficult to characterize; thus branched polyesters provide challanging test for TOF-SIMS. TOF-SIMS spectra of polyesters are obtained from thin polymer films cast from solution on a silver substrate. A good solvent for a polumer solution disrupts intermolecular forces between polymer chains but leaves the polumer intact. Transesterification reactions are potentially useful for characterization of high molecular weight and intractable polyesters. Transesterification products of polyesters and trifluoroacetic acid or an integral number of polyester repeat units and an additional diol. The progress of such reactions was monitored using peak intensities of reactants and products in TOF-SIMS spectra. The increasing abundance of tagged ions indicates that the reaction has progressed with time.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.5
• /
• pp.845-852
• /
• 2001

A microorganism producing fibrinolytic enzyme was isolated from Korean traditional soybean paste and identified as Bacillus sp. KDO-13. The fibrinolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-celluose, and gel chromatography on Sephadex G-100 of the culture supernatant of Bacillus sp. KDO-13. The molecular weight of the purified enzyme was estimated to be 44,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 8.0 and $50{\circ}C$, respectively. The enzyme activity was relatively stable at pH 7.0-9.0 and temperature below $50{\circ}C$. the activity of the enzyme was inhibited by $AI^{3+}$ and $Hg^{2+}$, but activated by $Co^{2+}$\;and\;Ni^{2+}. In addition, the enzyme activity was potently inhibited by EDTA and 0-phenanthroline. The purified enzyme could completely hydrolyze a fibrin substrate within 6 h in vitro, and had a low $K_m$ value for fibrin hydrolysis. It was concluded that the purified enzyme was a metalloprotease with relatively high specificity for fibrinolysis, and thus, could be applied as an effective thrombolytic agent.

• BMB Reports
• /
• v.37 no.6
• /
• pp.684-690
• /
• 2004

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and $50^{\circ}C$. It was rather stable within the range of 25 to $50^{\circ}C$ but lost activity rapidly above $50^{\circ}C$. The enzyme was activated by $Co^{2+}$ or EDTA and stabilized by dithiothreitol. The kinetic constants, $K_m$ and $V_{max}$ for heparin were $1.3{\times}10^{-5}\;M$ and $8.8\;{\mu}mol/min{\cdot}mg$. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

• Korean Journal of Biological Psychiatry
• /
• v.8 no.2
• /
• pp.239-245
• /
• 2001

Objective : Dopamine transporter is member of family of Na/Cl dependent neurotransmitter transporter, 12 transmembrane domain, that has high substrate specificity, affinity. It is related with dopamine reuptake in presynaptic vesicle. DAT has a VNTR in its 3'-untranslated region(UTR). 3'-UTR VNTR polymorphism is related with modification of dopamine transmission. The association between with VNTR polymorphism and neuropsychiatric disorders such as alcohol dependence, and low activity ALDH has been studied, but their relationship is unclear. We study about association of 3'-UTR VNTR of DAT gene and G2319A and alcohol dependence. Method : Group of Korean subjects were studied with alcohol dependence(n=49 male) compared to mentally healthy controls(n=53 male). The peripheral blood sample was acquired, and Polymerase Chain Reaction(PCR) amplification, MspI procedure was done. Result : There was a significant difference between alcohol dependence group and normal control(genotype frequency p<0.05, allele frequency p<0.05) Allele A frequency and genotype(GG, GA) frequency was a significant difference between alcohol dependence group and normal control(p<0.05). Conclusion : Our study showed that genetic polymorphism of DAT1 G2319A had relation with alcohol dependence.

• The Korean Journal of Mycology
• /
• v.36 no.2
• /
• pp.183-188
• /
• 2008

A fibrinolytic serine protease was purified from the fruiting bodies of an edible mushroom, Albatrellus confluens. The enzyme had a molecular mass of 30086.41 Da, as measured by MALDI-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Glu-Thr-Val-Thr-Glu-Thr-Thr-Ala -Pro-Trp-Gly-Leu-Ser-Arg-Ile. It displayed optimal activity at $50^{\circ}C$ and within a pH range of $8.0{\sim}10.0$, suggesting that the enzyme is an alkaline protease. The enzyme was stable up to $30^{\circ}C$. The enzyme displayed a strong substrate specificity for the synthetic peptide, N-Suc-Ala-Ala-Pro-Phe pNA. The enzyme activity was completely inhibited by addition of PMSF, indicating that the enzyme is a serine protease. No inhibition was observed following addition of E-64, pepstatin, or EDTA. The activity of the purified enzyme was decreased in the presence $Fe^{2+}$ or $Co^{2+}$, and the enzyme was completely inhibited by addition of $Hg^{2+}$. From these results, we propose that Albatrellus confluens could be used for biofunctional foods development and has potential therapeutic value for the treatment of vascular diseases.

• 한국생물공학회:학술대회논문집
• /
• 2002.04a
• /
• pp.457-460
• /
• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.