• Current Research on Agriculture and Life Sciences
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• v.4
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• pp.55-64
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• 1986

Polyphenol oxidase in japanese pear (Pyrus communis var. mansamkil) was isolated, partially purified and its some properties were investigated. Polyacrylamide disc gel electrophoresis indicated two bands with polyphenol oxidase activity in the extract from acetone dry powder of par flesh. These two polyphenol oxidases (PPO A and PPO B) were purified through acetone precipitation and diethylaminoethyl cellulose column chromatography. PPO A and B were purified 7.8 fold and 8.7 fold by the present procedure, respectively. The Rm values of partially purified PPO A and B were estimated to be 0.58 and 0.68, respectively. The optimum temp, and pH of PPO A activity were $33^{\circ}C$ and pH 7.0, while those of PPO B were $30^{\circ}C$ and pH 4.2, respectively. Two PPO were unstable over the temperature of $60^{\circ}C$. The substrate specificity of pear PPO showed high affinity toward o-diphenolic compounds, especially catechol in PPO A and chlorogenic acid in PPO B, but inactive toward m-diphenol, p-diphenol and monophenols. PPO A showed affinity toward the trihydroxyphenolic compound. $Zn^{{+}{+}}$ activated the PPO A activity but $Fe^{{+}{+}}$ inhibited PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity but $K^+$, $Mg^{{+}{+}}$, $Ca^{{+}{+}}$ and $Hg^{{+}{+}}$ inhibited at 10mM concentration. $Cu^{{+}{+}}$ activated the enzyme action at low concentrations but inhibited at high concentration. Inhibition studies indicated that L-ascorbic acid, L-cysteine and thiourea were most potent. The Km values of PPO A and PPO B for catechol were 20mM and 14.3mM, respectively.

• Journal of Life Science
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• v.29 no.4
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• pp.492-498
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• 2019

Previously, three kinds of lipases, lipAD1, lipAD2, and lipAD3, and lipase chaperone, lipBD, of Acinetobacter schindleri DYL129 isolated from soil sample were reported. In this report, three lipase and lipase chaperone were cloned into the pET32a(+) or pGEX-6P-1 vectors for protein expression in Escherichia coli, and named as pETLAD1, pETLAD2, pETLAD3 and pETLBD or pGEXLAD1, pGEXLAD 2, pGEXLAD3 and pGEXLBD, respectively. Protein expression rate was higher in pET system than in pGEX system. Although LipAD1 and LipAD2 were produced as inclusion bodies, their expression levels were high. So LipAD1 and LipAD2 could be solubilized in 8 M urea buffer and purified. LipAD3 and LipBD were overexpressed in soluble form and purified. Those proteins were purified by His-tag affinity chromatography connected in AKTA prime system. The activities of the purified lipases were demonstrated with 1% tributyrin agar plate. After purification, molecular mass was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LipAD1 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl butyrate, LipAD2 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl myristate, and LipAD3 showed high activity toward ${\rho}$-nitrophenyl acetate, ${\rho}$-nitrophenyl butyrate, and ${\rho}$-nitrophenyl miristate, respectively. Three lipases, LipAD1, LipAD2, and LipAD3, showed optimal reaction at $50^{\circ}C$ using ${\rho}$-nitrophenyl butyrate, as substrate.

• Journal of Life Science
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• v.32 no.2
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• pp.135-141
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• 2022

Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.15 no.2
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• pp.123-136
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• 1982

We investigated the changes in protein and free amino acid compositions of the muscles, and amino acid composition of the muscle proteins during postmortem storage of dorsal white and lateral dark muscles of Yellowtail, Seriola quinqueradita, which were kept at $2^{\circ}C$. We present an extensive discussion on the relationship between the changes of freshness and those of protein compositions in the white and the dark muscle of the red-fleshed fish by analyzing polyacrylamide gel electrophoretograms of $NaDodSO_4-solubilized$ sarcoplasmic and myofibrillar proteins extracted from the both muscles. By assessing K-value, total volatile basic nitrogen and pH value as a criterion of freshness, we found that the dark muscle undergoes a more rapid decrease in its freshness compared to that of the white muscle. The contents of the sarcoplasmic and the myofibrillar protein were decreased with postmortem aging of the muscles while those of the residual intracellular protein were increased, and these changes were somewhat faster in the dark muscle than in the white muscle. From the analysis of the electrophoretograms and their densitograms, we found that the sarcoplasmic proteins of the white and the dark muscle were respectively composed of 16 and 12 components. The sarcoplasmic protein of the white muscle lapsed for 10 days showed an increase of 18,000 and 41,000 dalton components, and a gradual decrease of 23,000 and 23,500 dalton components, whereas the sarcoplasmic protein of the dark muscle lapsed for 9 days showed a decrease of 49,000 dalton component, an appearence of a newly formed component of 47,000 dalton, and a disappearance of 26,000 dalton component. The electrophoretograms of the myofibrillar proteins shelved that the white and the dark muscle were composed of 17 and 16 components, respectively. Depending on the lapsed time of postmortem under the controlled condition, the myofibrillar proteins of the white muscle showed an increase of 40,000 dalton component, a gradual decrease of 37,500 dalton component, an appearance of a newly forming component of 32,000 dalton and a disappearance of 26,000 dalton component. On the other hand, the myofibrillar proteins of the dark muscle showed an increase of 58,000 and 64,000 dalton bands, a disappearance of light chain-2 protein and an appearance of a newly forming protein of 32,000 dalton. These changes on the electrophoretic patterns in the dark muscle were more rapid than those in the white muscle. In almost all of the cases, we observed that the changes in the sarcoplasmic protein were faster than those in the myofibrillar protein. The analysis of amino acid of the both muscle proteins showed that the white muscle was rich in glutamic acid, aspartic acid, leucine, arginine, lysine, etc. but was poor in proline and tryptophan. No significant difference was found in the amino acid composition of protein of both the white and the dark muscles. The sample of white muscle lapsed for 10 days shows a remarkable decrease in glutamic and aspartic acids, while that of the dark muscle lapsed for 9 days shows an appreciable decrease in alanine, glycine and arginine. The free amino acid compositions of the white and the dark muscles are respectively characterized with $63\%$ of histidine and $67\%$ of taurine with respect to the total free amino acids of the yellowtail at-death, respectively. The white muscle lapsed for 10 days showed an increase of histidine, valine and taurine, and a slight decrease of alanine, leucine and glycine. The dark muscle lapsed for 9 days shelved an increase of taurine, phenylalanine and glycine, and a decrease of histidine, alanine and serine.

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.577-598
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• 1996

This study was conducted to identify the effects of caffeine or combinations of caffeine and iron or vitamin E on the lipid and protein components or blood chemistry levels of the serum as well as the total homogenate, mitochondrial and microsomal fraction of the rat(Sprague-Dawley, female) liver. Chronic test were conducted to determine those effects. The chronic test was conducted by dividing rats into 5 groups according to the type of drugs and dosages administrated as follows; the control(group A), and group B was given 25mg/kg caffeine orally once daily for 30 days, group C was given 50mg/kg caffeine orally once daily for 30 days, group D was given 25mg/kg caffeine and orally ferric chloride once daily for 30 days and group E was given 25mg/kg caffeine and 25mg/kg vitamin E once daily for 30 days. The concentrations of glucose, urea nitrogen, uric acid, creatinine, total protein, albumin, A/G ratio, triglyceride, total cholesterol, HDL-cholesterol, free fatty acid, phospholipid as well as the activities of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP) were measured in the serum of each experimental groups. The concentrations of the carbonyl group and malondiaidehyde(MDA) and the patterns of the SDS-PAGE(Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) and fatty acid compositions in free fatty acids and phospholipids were analyzed to determine the oxidative damages and metabolic changes on the lipid and protein components in the serum, and total homogenate, mitochondrial and microsomal fractions of the rat liver. The results obtained from this study were summarized as follows; 1. Body weights of groups B, C, D and E were significantly decreased(p < 0.01) in comparison with that of the control in the chronic test. 2. The concentrations of serum glucose in groups B(124.5mg/dl), C(130.1mg/dl), D(122.1mg/dl), E(119.3mg/dl) were significantly higher(p < 0.01) in comparison to that of the control(101.5mg/dl). But, there were no significant differences in the concentrations of urea nitrogen, uric acid, creatinine, total protein, albumin and A/G ratio in comparison to that of the control. 3. The concentrations of total cholesterol and HDL-cholesterol in serum of groups B(69.6, 53.4mg/dl), C(73.0, 56.3mg/dl), D(68.9, 51.1mg/dl) and E(68.2, 51.3mg/dl) were significantly higher(p < 0.01) in comparison to that of the control(52.6, 38.8mg/dl). On the other hand, the concentrations of triglyceride in serum of groups B(45.0mg/dl), C(40.4mg/dl), D(33.8mg/dl) and E(47.2mg/dl) were significantly lower(p < 0.01) in comparison to that of the control(66.2mg/dl). There were no significant differences in the activities of ALT, AST and ALP in comparison to that of the control. 4. The concentrations of free fatty acid and phospholipid in serum of groups B(45.7, 154.4mg/dl), C(50.0, 167.2mg/dl), D(52.5, 148.4mg/dl) and E(41.1, 159.2mg/dl) were higher(p < 0.01) in comparison to that of the control(35.2, 125.3mg/dl). And the concentrations of the carbonyl group and malondialdehyde in serum of group D(1.82, 0.52nM/mg protein) were significantly higher(p < 0.01) in comparison to the control(1.53nM/mg protein). 5. The concentrations of carbonyl group in total homogenate, mitochondrial and microsomal fraction of group D(1.45, 0.94, 1.67nM/mg protein) were significantly higher (p < 0.01) in comparison to the control(1.16, 0.66, 1.27nM/mg protein). And the concentrations of malondialdehyde in the total homogenate, mitochondrial and microsomal fraction of group D(6.70, 6.10, 1.36nM/mg protein) were significantly higher(p < 0.01) in comparison to the control(5.17, 3.64, 0.68nM/mg protein). 6. As the analytical results of the fatty acid compositions of free fatty acid in serum, the proportions of stearic acid and arachidonic acid of groups B(16.52, 12.62%), C(17.52, 15.18%), D(19.73, 13.47%) and E(17.62, 13.28%) were significantly higher(p < 0.01) in comparison to the control(14.75, 7.88%), but the proportions of oleic acid and linoleic acid of groups B(12.97, 32.59%), C(10.88, 31.23%), D(12.37, 30.66%) and E(11.95, 32.41%) were significantly lower(p < 0.01) in comparison to the control(16.44, 35.12%). Otherwise, as the results of the fatty acid compositions of phospholipid in serum, the proportions of stearic acid and arachidonic acid of groups B(39.37, 16.39%), C(40.63, 17.83%), D(42.73, 15.39%) and E(39.16, 15.70%) were significantly higher(p < 0.01) in comparison to the control(37.74, 14.24%), but the proportions of oleic acid and linoleic acid of groups B(4.03, 14.38%), C(3.54, 12.38%), D(4.52, 11.68%) and E(4.29, 13.64%) were significantly lower(p < 0.01) in comparison to the control(5.53, 16.14%). 7. As the analytical results of the fatty acid compositions of free fatty acid in total homogenate, mitochondrial and microsomal fraction of liver, the proportions of oleic acid of groups B(7.8**, 8.73**, 6.88%) and C(6.89**, 7.75**, 6.58%) were lower(**:p < 0.01) in comparison to the control(8.67, 10.08, 7.81%), but the proportions of arachidonic acid of group C(22.62, 19.79, 23.71%) were significantly higher(p < 0.01) in comparison to the control(20.93, 18.47, 22.24%). And the proportions of palmitic acid of group D(25.95**, 26.16, 26.34**%) were significantly higher(**:p < 0.01) in comparison to the control(24.43, 25.42, 23.34%). In addition, the proportions of linoleic acid of group D(23.43, 25.02, 23.95%) were also significantly higher(p < 0.01) in comparison to the control(22.17, 23.75, 21.26%). The proportions of stearic acid of group D(19.87, 19.76**%) in mitochondrial and microsomal fraction were lower(**:p < 0.01) in comparison to the control(21.01, 24.18%), and the proportions of stearic acid of group E(16.71*, 19.65**%) in mitochondrial and microsomal fraction were significantly lower(**:p < 0.01, *:p < 0.05) in comparison to the control(21.01, 24.18%), and the proportions of linoleic acid of group E(25.04, 29.20, 26.48%) in total homogenate, mitochondria and microsome were significantly higher(p < 0.01) in comparison to the control(22.17, 23.75, 21.26%). 8. As the results of the fatty acid compositions of phospholipid in total homogenate, mitochondrial and microsomal fraction of liver, the proportions of palmitic acid of group D(17.58**, 18.78*, 18.23%**) were significantly higher(**:p < 0.01, *:p < 0.05) in comparison to the control(16.28, 17.22, 16.38%), and the proportions of stearic acid of group D(36.41, 37.23, 39.53%) were also significantly higher(p < 0.01) in comparison to the control(34.18, 34.16, 36.04%). But the proportions of oleic acid(3.41*, 3.11**, 3.12**%) and linoleic acid (18.03**, 15.79**, 14.74**%) of group D were significantly lower(**:p < 0.01, *:p < 0.05) in comparison to the control(oleic : 3.63, 3.72, 3.79%, linoleic : 20.03, 18.71, 18.48%). 9. In order to determine the oxidative damages to the protein in serum, mitochondrial and microsomal fraction of the rat liver, the patterns of the SDS-PAGE were identified, but the results of SDS-PAGE were not significantly different between the control and experimental groups.

• Tuberculosis and Respiratory Diseases
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• v.45 no.6
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• pp.1236-1251
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• 1998

Background : TNF-$\alpha$ appears to be a central mediator of the host response to sepsis. While TNF-$\alpha$ is mainly considered a proinflammatory cytokine, it can also act as a direct cytotoxic cytokine. However, there are not so many studies about the relationship bet ween TNF-$\alpha$ level and lung injury severity in ALI, particularly regarding the case of ALI caused by direct lung injury such as diffuse pulmonary infection. Recently, a natural defense mechanism, known as the stress response or the heat shock response, has been reported in cellular or tissue injury reaction. There are a number of reports examining the protective role of pre-induced heat stress proteins on subsequent LPS-induced TNF-$\alpha$ release from monocyte or macrophage and also on subsequent LPS-induced ALI in animals. However it is not well established whether the stress protein synthesis such as HSP can be induced from rat alveolar macrophages by in vitro or in vivo LPS stimulation. Methods : We measured the level of TNF-$\alpha$, the percentage of inflammatory cells in bronchoalveolar lavage fluid, protein synthesis in alveolar macrophages isolated from rats at 1, 2, 3, 4, 6, 12, and 24 hours after intratracheal LPS instillation. We performed histologic examination and also obtained histologic lung injury index score in lungs from other rats at 1, 2, 3, 4, 6, 12, 24 h after intratracheal LPS instillation. Isolated non-stimulated macrophages were incubated for 2 h with different concentration of LPS (0, 1, 10, 100 ng/ml, 1, or 10 ${\mu}g/ml$). Other non-stimulated macrophages were exposed at $43^{\circ}C$ for 15 min, then returned to at $37^{\circ}C$ in 5% CO2-95% for 1 hour, and then incubated for 2 h with LPS (0, 1, 10, 100ng/ml, 1, or 10 ${\mu}g/ml$). Results : TNF-$\alpha$ levels began to increase significantly at 1 h, reached a peak at 3 h (P<0.0001), began to decrease at 6 h, and returned to control level at 12 h after LPS instillation. The percentage of inflammatory cells (neutrophils and alveolar macrophages) began to change significantly at 2 h, reached a peak at 6 h, began to recover but still showed significant change at 12 h, and showed insignificant change at 24 h after LPS instillation compared with the normal control. After LPS instillation, the score of histologic lung injury index reached a maximum value at 6 h and remained steady for 24 hours. 35 kDa protein band was newly synthesized in alveolar macrophage from 1 hour on for 24 hours after LPS instillation. Inducible heat stress protein 72 was not found in any alveolar macrophages obtained from rats after LPS instillation. TNF-$\alpha$ levels in supernatants of LPS-stimulated macro phages were significantly higher than those of non-stimulated macrophages(p<0.05). Following LPS stimulation, TNF-$\alpha$ levels in supernatants were significantly lower after heat treatment than in those without heat treatment (p<0.05). The inducible heat stress protein 72 was not found at any concentrations of LPS stimulation. Whereas the 35 kDa protein band was exclusively found at dose of LPS of 10 ${\mu}g/ml$. Conclusion : TNF-$\alpha$ has a direct or indirect close relationship with lung injury severity in acute lung injury or acute respiratory distress syndrome. In vivo and in vitro LPS stimulation dose not induce heat stress protein 72 in alveolar macrophages. It is likely that 35 kDa protein, synthesized by alveolar macrophage after LPS instillation, does not have a defense role in acute lung injury.