• Korean Journal of Microbiology
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• v.31 no.2
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• pp.148-156
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• 1993

Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were $ 2.0 * 10^{-4}$ M for inosine, $2.0 *10^{-3}$ M for deoxyinosine, $ 2.0 * 10^{-5}$ M for guanosine and $2.0 10 ^{-5}$ M for deoxyguanosine. According to the ratio of relative $K_{cat}$Km, substrate specificity of each nucleoside was in the order of guanosine or deoxyguanosine, inosine and deoxyinosine. Cosubstrate, phosphate, revealed downward curvature in Lineweaver-Burk plot at high concentrations, indicating a negative cooperativity between subunits. The inhibition constants for purine analogs were measured to be $ 6 * 10^{-4}$ M for formycin B as the competitive inhibitor of inosine, $ 9 * 10^{-6}$ M for guanine as the competitive inhibitor of guanosine, $2 * 10^{-4}$ M for hypoxanthine as the non competitive inhibitor of guanosine and $4.5 * 10 ^{-4}$ M for 6-mercaptopurine as the non competitive inhibitor of guanosine. Alternative substrates, guanosine, deoxyguanosine and adenosine were found to act as competitive inhibitors with Ki values o $f^ 2.0 * 10 {-5}$ M, $2.6 * 10^{-5}$ M and $8.5 * 10 ^{-4}$ M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of $1.8 * 10^{-5}$ M and $ 3.0 * 10^{-5}$ M, respectively, when deoxyinesine was the variable substrate. The results of alternative substrate sstudies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine.e.

• Journal of Chest Surgery
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• v.46 no.1
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• pp.14-21
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• 2013

Background: Reactive oxygen species (ROS) are known to be related to cardiovascular diseases. Many studies have demonstrated that angiotensin-converting enzyme inhibitors have beneficial effects against ROS. We investigated the antioxidant effect of captopril and enalapril in nitric oxide mediated vascular endothelium-dependent relaxations. Materials and Methods: Isolated rabbit abdominal aorta ring segments were exposed to ROS by electrolysis of the organ bath medium (Krebs-Henseleit solution) after pretreatment with various concentrations (range, $10^{-5}$ to $3{\times}10^{-4}$ M) of captopril and enalapril. Before and after electrolysis, the endothelial function was measured by preconstricting the vessels with norepinephrine ($10^{-6}$ M) followed by the cumulative addition of acetylcholine (range, $3{\times}10^{-8}$ to $10^{-6}$ M). The relevance of the superoxide anion and hydrogen peroxide scavenging effect of captopril and enalapril was investigated using additional pretreatments of diethyldithiocarbamate (DETCA, 0.5 mM), an inhibitor of Cu/Zn superoxide dismutase, and 3-amino-1,2,4-triazole (3AT, 50 mM), an inhibitor of catalase. Results: Both captopril and enalapril preserved vascular endothelium-dependent relaxation after exposure to ROS in a dose-dependent manner (p<0.0001). Pretreatment with DETCA attenuated the antioxidant effect of captopril and enalapril (p<0.0001), but pretreatment with 3AT did not have an effect. Conclusion: Both captopril and enalapril protect endothelium against ROS in a dose-dependent fashion in isolated rabbit abdominal aortas. This protective effect is related to superoxide anion scavenging.

• Journal of the Korean Chemical Society
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• v.41 no.6
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• pp.304-312
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• 1997

Acetolactate synthase(ALS) is the common enzyme in the biosynthetic of valine, leucine, and isoleucine, and is the target of several classes of structually unrelated herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. In an effort to develop new and desirable herbicides, we have synthesized 4,6-dimethoxypyrimidine derivatives, and examined their inhibitory activities on pea ALS. The most active compound was found to be K11570 and $IC_{50}$ value for K11570 was 0.2 ${\mu}M.$ The inhibition of pea ALS by K11570 was biphasic, showing increased inhibition with incubation time. The K11570 showed mixed-type inhibition with respect to substrate pyruvate. Dual inhibition analysis of K11570 versus sufonylurea herbicide Ally and feedback inhibitor leucine revealed that three inhibitors were competitive for binding to ALS. The arginine modified enzyme showed decreased inhibition by K11570, sufonylurea Ally, and leucine, in constrast to, tryptophan modification did not affect on the sensitivity of ALS to the inhibitors.

• Korean Journal of Food Science and Technology
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• v.16 no.4
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• pp.392-397
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• 1984

These experiments were conducted to investigate general enzymatic characteristics of two forms(glucoamylase I and glucoamylase II) of the purified glucoamylase produced by Rhizopus oryzae. Molecular weights of glucoamylase I and glucoamylase II estimated by Sephadex G-100gel filtration, were approximately 101,000 and 115,000, respectively, and those estimated by SDS-polyacrylamide gel electrophoresis being 120,000 and 127,000, respectively. Isoelectric points of the above enzyme were pH 7.25 and pH 7.75. The optimum temperature was $50^{\circ}C$ and the enzyme was stable below $45^{\circ}C$. Optimum pH of both glucoamylase I and glucoamylase II was about pH 5.0. The stable pH range of them were pH 3.5-8.0 and 4.5-8.0, respectively. Michaelis constants of glucoamylase I and glucoamylase II toward souluble starch were 4.545 mg/ml and 5.560 mg/ml, respectively. $Hg^{++}$, $Pb^{++}$, p-CMB and IAA were inhibitors of glucoamylase I and $Hg^{++}$, $Mn^{++}$, p-CMB and IAA were inhibitors of glucoamylase II.

• Journal of Applied Biological Chemistry
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• v.63 no.3
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• pp.181-188
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• 2020

In this study, Tongue fern (Pyrrosia lingua) plants that have been used traditionally as medicines. Their traditional medicinal uses, regions where indigenous people use the plants, parts of the plants used as medicines. This study was designed to assess the antioxidant and inhibition activities of extracts from P. lingua. In the P. lingua extracts was measured ethanol activity, 80.0% ethanol was high activity. The antioxidant activity was measured in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), assays. DPPH and ABTS radical in this experiment, solid and phenolic of extract were tested, but only an average concentration of 100 ㎍/mL was used. However, the phenolic extract is shown phenolic activity reached a peak. Also, phenolic extracts ware reached peak water and ethanol extracts. As a result, using the phenolic extracts did other antioxidant assays such as DPPH, ABTS, protection factor, and thiobarbituric acid reactive substances at 50-200 ㎍/mL concentrations. The activity of elastase and collagenase, inhibiting their activities may retard skin aging. α-Glucosidase and α-amylase, inhibitors need to be explored for the benefit of postprandial hyperglycemia in diabetic patients. Activities of tyrosinase, hyaluronidase and xanthine oxidase inhibitors of these enzymes are increasingly important ingredients in cosmetics and medications to protect the skin against hyperpigmentation and skin aging. Inhibition effects were investigated using the P. lingua extracts at 50-200 ㎍/mL concentrations. The expression levels of enzyme inhibitions activities were decrease in dependent-concentrations manner when P. lingua extracts were treated.

• Animal Bioscience
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• v.36 no.10
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• pp.1604-1611
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• 2023

Objective: The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Methods: Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Results: Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dose-dependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Conclusion: Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

• Journal of Pharmaceutical Investigation
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• v.26 no.3
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• pp.175-185
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• 1996

To inhibit the enzymatic degradation of leucine enkephalin (Leu-Enk) and its synthetic analog. $[D-ala^2]$-leucine enkephalinamide (YAGFL), in the nasal, rectal and vaginal mucosal and serosal extracts of rabbits, effects of enzyme inhibitors such as amastatin (AM), puromycin (PM), thiorphan (TP), thimerosal (TM), EDTA, N-carboxymethyl-Phe-Leu (CPL), phenylethyl alcohol (PEA), phenylmercuric acetate (PMA), benzalkonium chloride (BC) and modified cyclodextrins, alone or in combination, were observed by assaying the pentapeptides staying intact during incubation. Mucosa extracts were prepared by exposing freshly-excised mucosal specimens mounted on Valia-Chien cells to isotonic phosphate buffer while stirring. The degradation of Leu-Enk and YAGFL followed the apparent first-order kinetics. The half-lives (mean) in the nasal, rectal and vaginal mucosal extracts were found to be 1.07, 0.33 and 1.14 hr for Leu-Enk, and 16.9, 6.2 and 6.8 hr for YAGFL, respectively. AM or PM, which is an aminopeptidase inhibitor, did not show a sufficient inhibition of Leu-Enk $(50\;{\mu}g/ml)$ degradation in all kinds of extracts. $Dimethyl-{\beta}-cyclodextrin\;(DM-{\beta}-CyD)$ decreased the degradation rate constants of Leu-Enk about 2 or 3 times, comparing with no additive. However, the use of mixed inhibitors of AM $(50\;{\mu}M)$/TM (0.25 mM)/EDTA (5 mM) resulted in a full stabilization of Leu-Enk by decreasing the degradation rate constants 67.3, 161.3 and 113.8 times far the nasal, rectal and vaginal mucosal extracts, respectively, comparing with no inhibitor. With mixed inhibitors, Leu-Enk remained intact more than 90% after 6 hr-incubation. In the stabilization of YAGFL, hM, TP or CPL alone showed little efffct, and some additives demonstrated a considerable inhibition of YAGFL degradation in the rank order of TM > BC > EDTA. However, the addition of mixed inhibitors such as TM (0.5 mM) and EDTA (5 mM) into the extracts protected YAGFL from the degradation by more than 85% even after 24 hr-incubation, suggesting almost complete inhibition of YAGFL degradation in the extract. On the other hand, $DM-{\beta}-CyD\;or\;hydroxypropyl-{\beta}-cyclodextrin$ (10%) were also found to retard enzymatic degradation rates of YAGFL markedly, and resulted in staying intact more than 80% of YAGFL in the nasal and vaginal mucosal extracts, and more than 60% in the rectal mucosal extract after 16 hr-incubation.

• Applied Biological Chemistry
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• v.35 no.4
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• pp.237-241
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• 1992

The protease from Halomonas sp. ES 10 was purified by methanol precipitation, gel filtration on Sephadex G-150 and G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The specific activity of purified enzyme was 1,014 units/mg protein, and the yield of the total activity from the culture filtrate was 7%. The optimal temperature and pH for the enzyme activity were $35^{\circ}C$, and pH 11.0, respectively. And the enzyme was stable in the range of $pH\;7.5{\sim}11.0$. The residual activity of the enzyme was 70%, when the enzyme was incubated at $50^{\circ}C$ for 40 min. The Km value of the enzyme was 7.4 mg/ml to milk casein. $Li^+$, $Ca^{2+}$, SDS and Tween 80 were appeared to activators, whereas $Hg^{2+}$ and EDTA to inhibitors. The addition of DFP and PMSF showed the relative enzyme activities of 63% and 107%, respectively, suggesting that the enzyme may not belong to serine type protease. When the alkaline protease was treated with 0.5 M and 1 M NaCl, the relative enzyme activities were 95% and 65%, respectively. This enzyme showed 20% and 15% higher enzyme activity than that of Aspergillus oryzae (Sigma Chemical Company product, P4755) in the presence of 0.5 M and 1 M NaCl.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.445-450
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• 1996

Gelatinase A is a member of the matrix metalloproteinases that play an important role in cancer invasion and metastasis. In the course of screening gelatinase A inhibitors from microbial sources, a fungal strain PT-262 showed a strong inhibitory activity. The strain was identified as Chaunopycnis alba on the basis of its morphological characteristics. The inhibitor was isolated from acetone extract of mycelial cake by sequential chromatographies on MCI-gel, Sephadex LH-20, and a reverse-phase HPLC column. The purified inhibitor was identified as pyridoxatin by its physico-chemical properties and spectroscopic analysis. Pyridoxatin is not a peptide analog and has cyclic hydroxamic acid moiety. It inhibited activated gelatinase A with an $IC_{50}$ value of 15.2 ${\mu}M$ using fluorescent synthetic peptide. It also had a strong cytotoxicity against human cancer cell lines in vitro. Furthermore, this compound inhibited DNA synthesis with an $IC_{50}$ value of 2.92 ${\mu}M$ in PC-3 prostate cancer cells by [$^3H$]thymidine incorporation assay.

• Journal of Life Science
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• v.7 no.3
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• pp.228-233
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• 1997

A thiol protease has been isolated and partially purified from encysted brine shrimp Artemia franciscana using a four-step procedure(filtration, salting out, gel filtration and ion exchange chromatography). The optimum pH of the enzyme for caseinolytic activity was appeared to be 3.0, and the enzymematic activity was stable up to pH 6.0 but lost completely at the pH higher than 8.0. The optimal temperature of the enzyme was appeared to be 35$^{\circ}$C, and ninety percent of the enzyme activity was lost at 45$^{\circ}$C. Various metal ions, e.g., zinc, copper, iron, inhibited the enzyme activity; however, heavy metal chelator, e.g., EDTA, stimulated the enzyme activity. The protease was concluded to be a member of the thiol group protease, since it was inhibited by thiol protease inhibitors and iodoacetate. The protease was also concluded to be a acid protease based on optimum pH.