• YAKHAK HOEJI
• /
• v.44 no.2
• /
• pp.197-203
• /
• 2000

The enzymatic properties of $\beta$-galactosidases from Aspergillus oryzae, bovine liver and Saccharomyces pragilis have been studied using enzyme-linked lectin assay based on the RC $A_{120}$ and BS-II lectins which specifically bind to terminal galactose and GlcNAc residue, respectively. Asialofetuin, a monomeric glycoprotein with approximately 48 kDa in molecular weight, was used as a substrate. This glycoprotein contains three N-linked triantennary complex type carbohydrate chains with each of which terminating in Ga1$\beta$P1 longrightarrow4G1cNAc (74%). Their optimal pHs were 3.5 and 6.5 (A. oryzae), and 3.5~5.5 (bovine liver and S. fragilis) at 37$^{\circ}C$ during 24 hrs, and the effective concentrations were 0.9, 2.9, and 1.7 mg/ml, respectively The enzyme from A oryzae requires 100 mM N $a^{+}$ or $K^{+}$, while the enzyme from bovine liver requires $Ba^{2+}$ for activity. However all of the three $\beta$-galactosidases were inactivated by SDS and C $u^{2+}$. These results indicate that the hydrolysis of glycoprotein such as asialofetuin depends on the reaction conditions of $\beta$-galactosidases and some metal ions. ions.

• Korean Journal of Agricultural Science
• /
• v.21 no.2
• /
• pp.142-147
• /
• 1994

Characteristics of TNP-cellulose which prepared from carboxymethyl cellulose powder, CM32, as substrate for cellulase activity assay were investigated. Enzymatic hydrolysis of TNP-cellulose occured on the cellulose moiety but not on amide bonds, following Michaelis-Menten kinetics. Three cellulase preparations from Trichoderma viride, Aspergillus niger, and Cellulomonas sp. were tested for their pH and temperature dependences and compared with the method determining the increase in reducing power. The enzyme activity was found to have the same temperature range in both methods, however the pH range was broadened in the case of using TNP-cellulose as substrate. The colorimetric method for cellulase assay using TNP-cellulose as substrate was compared with the other methods: one based on determination of the increase in reducing power; and the other based on determining the decrease in viscosity of Na-CM-cellulose solution. The activities measured by the colorimetric method showed a linear correlation with the enzyme concentration of certain range in all three enzymes tested, and the activity values were proportional to those obtained from the other methods. Depending on the enzyme, however, the activity values from this method were not always in proportion to those from the viscometric method. suggesting that this method was not specific for determination of the endo-type cellulase.

• Journal of Life Science
• /
• v.29 no.3
• /
• pp.287-294
• /
• 2019

Calystegia soldanella is distributed in coastal sand dunes and has high environmental adaptability; it is also known to be effective for anti-oxidant, anti-pyretic, anti-septic, and diuretic action. This study investigated the effect of crude extracts and organic solvent fractions of C. soldanella on MMP-2 and MMP-9 expression, MMP activity, and cell mobility in phorbol-12-myristate-13-acetate (PMA)-induced fibrosarcoma HT-1080 cells. C. soldanella was twice extracted, once with methylene chloride (MC) and once with methanol (MeOH). After the MC and MeOH extracts were combined, their suppressive effects on MMP-2 and MMP-9 expression, MMP enzymatic activity, and gene and protein expression were measured by gelatin zymography, enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and western blot method. Cell mobility for the HT-1080 cells was observed by wound healing assay. The combined crude extracts showed a significant suppressive effects on MMP-2 and MMP-9 expression. To explore active inhibitory elements, the combined extracts were fractionated according to polarity into with n-hexane, 85% aqueous methanol, n-butanol, and water. Across these four solvent fractions, MMP-2 and MMP-9 activity and cell mobility in the HT-1080 cells were all strongly inhibited by the n-hexane fraction. These results suggest that C. soldanella extract and organic solvent fractions could be used as potent MMP inhibitors for effective anti-cancer treatments to suppress cancer invasion and metastasis.

• Fisheries and Aquatic Sciences
• /
• v.3 no.3_4
• /
• pp.195-199
• /
• 2000

Tyrosinase inhibitory activities of 14 kinds of seaweed, Ecklonia stolonifera, Ecklonia cava, Undaria pinnatiflda, Laminaria japonica, Sargassum fulvellum, Sargassum miyabei, Sargassum thunbergii, Porphyra yezoensis, Gracilaria verrucosa, Carpopeltis affinis, Pachymeniopsis elliptica, Gelidium amansii, Codium fragile and Ulva pertusa were determined using commercially available mushroom tyrosinase in an in vitro assay system. The $1\%$ (w/v) methanol extract from E. stolonifera showed the highest tyrosinase inhibitory activity of $79.0\%$, electron donating activity of $79.0\%$ and total phenol content of 3.75 mg/100g. Ethyl acetate-methanol-water (7 : 2 : 0.2, v/v) fraction $(0.5\%,\;w/v)$ isolated from the methanol extract showed tyrosinase inhibitory activity of $75.9 \%$, electron donating activity of $88.1 \%$ and total phenol content of 4.38 mg/100g. Tyrosinase inhibitory activity was closely associated with total phenol content (R = 0.99) and electron donating activity (R=0.99). Maximum absorption wavelength of the fraction was 218nm and that of phenolic compounds showed about a range from 210 to 220nm. The inhibition mode of the fraction was noncompetitive inhibition.

• Ocean and Polar Research
• /
• v.41 no.2
• /
• pp.79-88
• /
• 2019

In this study, the inhibitory effect of Atriplex gmelinii C. A. Mey. against the activity of MMP-2 and MMP-9 secreted from phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 cells was evaluated by gelatin zymography and enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase-chain reaction (RT-PCR), and Western blot assay. Specimens of the halophyte A. gmelinii were extracted twice for 24 hr with methylene chloride ($CH_2Cl_2$), and then twice with methanol (MeOH), in turn. Each extract significantly inhibited the enzymatic activities in gelatin zymography and MMP ELISA kit, and expression of MMP-2 and 9 in mRNA and protein levels. Two crude extracts were combined and then the combined crude extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water ($H_2O$) fractions, according to solvent polarity. Among solvent-partitioned fractions, the 85% aq.MeOH fraction showed the strongest inhibitory effect against MMP-2 and -9 in gelatin zymography and MMP ELISA kit. In RT-PCR, all solvent-partitioned fractions significantly suppressed mRNA expression of MMP-2 and -9. On the other hand, in Western blot assay, all solvent-partitioned fractions except $H_2O$ significantly reduced expression levels of protein. HT 1080 cell migration was most significantly inhibited by the n-BuOH fraction followed by the 85% aq.MeOH and $H_2O$ fractions. These results suggest that A. gmelinii could be used as a potential source to inhibit tumor cell metastasis.

• Biomedical Science Letters
• /
• v.12 no.4
• /
• pp.419-425
• /
• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• Journal of Life Science
• /
• v.18 no.1
• /
• pp.96-102
• /
• 2008

Piceatannol is a polyphenol that is found in abundant quantities in grapes and wine. Although recent experimental data revealed the anti-cancer potency of piceatannol, the molecular mechanisms underlying the antileukemic activity have not yet been studied in detail. In the present study, we investigated further possible mechanisms by which piceatannol exerts its anti-proliferative action in cultured human leukemia U937 cells. Exposure of U937 cells to piceatannol resulted in growth inhibition and induction of apoptosis as measured by MTT assay and flow cytometry analysis, which was associated with S phase arrest of the cell cycle. Piceatannol treatment markedly inhibited the activity of telomerase, and the levels of human telomerase reverse transcriptase (hTERT) and telomerase-associated protein-1 (TEP-1), main determinants of the telomerase enzymatic activity, were progressively down-regulated by piceatannol treatment in a dose-dependent fashion. However, the levels of cyclooxygenases (COXs) expression and prostaglandin E2 (PGE2) release were not changed in piceatannol-treated U937 cells. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of piceatannol.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.17 no.1
• /
• pp.152-158
• /
• 2016

Glutamate is one of the major excitatory neurotransmitters in the central nervous system of vertebrates. Human GDH (hGDH) is the enzyme that regulates the glutamate metabolism and its expression is higher in the brains of schizophrenia patients than in normal subjects. This study examined the changes in the hGDH enzymatic activity caused by antipsychotic drugs (haloperidol, risperidone, (${\pm}$)-sulpride, chlopromazine hydrochloride, melperone, (${\pm}$)butaclamol, domperidone, clozapine) related to schizophrenia. First of all, hGDH isozymes (hGDH1, hGDH2) were synthesized by genetic recombination. As a result of the enzyme assay, haloperidol, (${\pm}$)-sulpride, melperone and clozapine had an inhibitory effect on the hGDH isozymes. In addition, haloperidol showed a non-competitive inhibition against the substrate, 2-oxoglutarate. In contrast, it showed an uncompetitive inhibition against another substrate, NADH. The inhibitory effect of haloperidol on hGDH2 was abolished by the presence of L-leucine, an allosteric effector of hGDH, but by not other antipsychotic drugs. These results revealed the inhibition of enzyme activity by psychotropic drugs in hGDH isoenzymes (hGDH1 and hGDH2) and the possibility that haloperidol may be used to regulate the GDH activity and glutamate concentration in the central nervous system.

• Herbal Formula Science
• /
• v.28 no.4
• /
• pp.327-337
• /
• 2020

Objective : Citri Unshius Pericarpium is the dried peel of mature fruit of Citrus unshiu Markovich and has been used to treat indigestion, vomiting, and removal of phlegm. This study investigated the hepatoprotective and antioxidant effect of CEE (Ethanol extract of Citri Unshius Pericarpium) in cadmium (CdCl₂)-treated HepG2 cells. Methods : Component analysis of Citri Unshius Pericarpium was analyzed by UPLC with C₁₈ column. Cell viability was determined by MTT assay. The enzyme activity of superoxide dismutase (SOD) and the level of reactive oxygen species (ROS) and reduced glutathione (GSH) were analyzed using commercially available kits. Results : Cadmium caused severe HepG2 cell death. Cadmium also increased ROS production, consistent with depletion of GSH and inhibition of the SOD enzyme. However, CEE treatment reduced cell death and relieved oxidative stress caused by cadmium toxicity. CEE lowered ROS levels and improved depletion of GSH levels. CEE also enhanced the enzymatic activity of SOD. In component analysis, hesperidin was the most abundant of the five marker compounds (Narigenin, Narigin, Narirutin, Hesperidin and Hesperidin), which assumes that hesperidin partially contributed to the antioxidant activity of CEE. Conclusion : These results suggested that CEE could be a potential substance to solve heavy metal-related health problems. In particular, inhibition of oxidative stress by CEE can be a way to treat liver damage caused by cadmium.

• BMB Reports
• /
• v.34 no.3
• /
• pp.219-224
• /
• 2001

To identify the antioxidative peptides in the gelatin hydrolysate of bovine skin, the gelatin was hydrolyzed with serial digestions in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. The second enzymatic hydrolysate (hydrolyzed with pronase E) was composed of peptides ranging from 1.5 to 4.5 kDa, and showed the highest antioxidative activity, as determined by the thiobarbituric acid method. Three different peptides were purified from the second hydrolysate using consecutive chromatographic methods. This included gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an octadecylsilane chloride column. The isolated peptides were composed of 9 or 10 amino acid residues. They are: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp (PI), Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly (PII), and Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp (PIII), as characterized by Edman degradation and fast-atom bombardment mass spectrometry. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability with a methylthiazol tetrazolium assay The results showed that PII had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by the addition of the peptide. These results suggest that the purified peptide, PII, from the gelatin hydrolysate of bovine skin is a natural antioxidant, which has potent antioxidative activity.