• Title/Summary/Keyword: Gel electrophoresis

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Microsatellite Alterations of Plasma DNA in Non Small Cell Lung Cancer (비소세포폐암 환자의 혈장 DNA를 이용한 Microsatellite 분석)

  • Kim, Kyu-Sik;Kim, Eun-Jung;Kim, Soo-Ock;Oh, In-Jae;Park, Chang-Min;Jeong, Ju-Yeon;Kim, Yu-Il;Lim, Sung-Chul;Park, Jong-Tae;Kim, Young-Chul
    • Tuberculosis and Respiratory Diseases
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    • v.58 no.4
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    • pp.352-358
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    • 2005
  • Microsatellites are short tandem repeated nucleotide sequences that are present throughout the human genome. Variations in the repeat number or a loss of heterozygosity around the microsatellites have been termed a microsatellite alteration (MA). A MA reflects the genetic instability caused by an impairment in the DNA mismatch repair system and is suggested to be a novel tumorigenic mechanism. A number of studies have reported that MA in the DNA extracted from the plasma occurs at varying frequencies among patients with a non-small cell lung carcinoma (NSCLC). The genomic DNA from 9 subjects with a non-small cell lung cancer (squamous cell cancer 6, adenocarcinoma 2, non-small cell lung cancer1) and 9 age matched non-cancer control subjects (AMC: tuberculosis 3, other inflammatory lung disease 6) and 12 normal control subjects (NC) were extracted from the peripheral blood leukocytes and plasma. Three microsatellite loci were amplified with the primers targeting the Gene Bank sequence D21S1245, D3S1300, and D3S1234. MA in the form of an allelic loss or a band shift was examined with 6% polyacrylamide gel electrophoresis and silver staining. None (0/12) of the NC subjects less than 40 years of age showed a MA in any of the three markers, while 88.9%(8/9) of the AMC above 40 showed a MA in at least one of the three markers (p<0.05). Sixty percent(6/10) of the control subjects with a smoking history showed a MA in one of the three markers, while 9.1%(1/11) of the control subjects without smoking history showed a MA (p<0.05). However, not only did 66.7%(6/9) of lung cancer patients show a MA in at least one of the three markers but so did 88.9%(8/21) of the AMC patients (p>0.05). In conclusion, a MA in the D21S1245, D3S1300, and D3S1234 loci using DNA extracted from the plasma was detected in 66.7% of lung cancer while no MA was found in the young non-smoking control subjects. However, many of the non-cancer control subjects (aged smokers) also showed a MA, which compromised the specificity of the MA analysis as a screening test. Therefore, a further study with a larger sample size will be needed.

Genetic Variations in Geographic Venus Clam(Gomphina aequilatera, Sowerby) Populations from Samcheok and Wonsan (삼척과 원산의 지리적 민들조개(Gomphina aequilatera, Sowerby) 집단의 유전적 변이)

  • Kim, Jong-Rae;Jung, Chang-Ho;Kim, Yong-Ho;Yoon, Jong-Man
    • Development and Reproduction
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    • v.10 no.4
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    • pp.227-238
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    • 2006
  • Genomic DNAs(gDNAs) were isolated from the venus clam(Gomphina aequilatera) from Samcheok(venus clam from Samcheok; VCS) and Wonsan(venus clam from Wonsan; VCW) located in the East Sea of the Korean Peninsula. The amplified products were generated by agarose gel electrophoresis(AGE) with oligonucleotides primer, detected by staining with ethidium bromide and viewed by ultraviolet ray. The seven arbitrarily selected primers BION-21, BION-23, BION-25, BION-27, BION-29, BION-31 and BION-33 generated the shared loci, polymorphic, and specific loci, with the molecular sizes ranging from 150 bp to 2,400 bp. In this study, 147 polymorphic loci(147/954 loci, 15.41%) in VCS population and 274(274/996 loci, 27.51%) in VCW population were generated with seven primers. These results suggest the genetic variation in VCW population is higher than in VCS population. Especially, the 700 bp bands generated by the primer BION-21 were identified commonly in two Gomphina populations, which identified populations and/or species. This specific primer was found to be useful in the identification of individuals and/or population, resulting from the different DNA polymorphism among individuals/species/population. Two Gomphina populations between the individual SAMCHEOK no. 03 and WONSAN no. 22 showed the longest genetic distance(0.696) in comparison with other individuals used. The complete linkage cluster analysis indicating three genetic groupings and dendrogram revealed close relationships among individual identities within two geographical populations of venus clam(G. aequilatera) from the Samcheok and Wonsan. The intra-species classification and clustering analyses inferred from molecular markers supported the traditional taxonomy of the species based on morphological characters such as shell size, shape and color. Accordingly, as mentioned above, RAPD analysis showed that VCS population was more or less separated from VCW population.

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Effect of Proteinase Activity on the Cheddar Cheese Quality (단백분해 효소 활성(蛋白分解 酵素 活性)이 Cheddar Cheese의 품질(品質)에 미치는 영향(影響))

  • Kim, Min-Bae
    • Journal of Dairy Science and Biotechnology
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    • v.14 no.2
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    • pp.157-164
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    • 1996
  • This study aimed increase the quality during ripening of Cheddar cheese made with proteinase-negative mutant of Streptococcus lactis KCTC 1913 selected by curing. The degradation of protein during cheese ripening were investigated by electrophoresis and chromatography. The results were summarized as follow ; 1. The number of lactic acid bacteria decreased with the ripening stage, and that of the control cheese decreased faster than that of the cheese made with mutant. 2. Polyacrylamide gel electrophoretic analysis of cheese caseins revealed no difference between the cheese made with mutant and the control cheese, but differences along with the ripening stage were evident. 3. On Sephadex G-25 column chromatography, the extracts of bitter components from the green cheese and 3 month ripended cheese were fractionated into 3 fractions. With the progress of ripening, bitter peptides were degraded to rather small peptides or free amino acids. 4. Sensory evaluation of the 3 month ripended Cheddar cheese found no significant differences in color but the cheese made with mutant evidenced higher palatability in flavor and better texture than the control cheese. 5. The yields of the cheddar cheese made with mutant was 0.14% higher than that of the control cheese.

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High Glucose Induces Apoptosis through Caspase-3 Dependent Pathway in Human Retinal Endothelial Cell Line (인간망막 내피세포주에서 고농도 포도당이 caspase-3 경로를 통해 세포자연사 유도)

  • Seo, Eun-Sun;Chae, Soo-Chul;Kho, Eun-Gyeong;Lee, Jong-Bin
    • Korean Journal of Environmental Biology
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    • v.27 no.1
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    • pp.66-72
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    • 2009
  • Diabetic Retinopathy (DR) is a leading cause of blindness among adults in the western countries. Hyperglycemia is a condition, that induces apoptotic cell death in a variety of cell types in diabetes, but the mechanism remains unclear. The aim of the study is to understand the effects of high Glucose on Human Retinal Endothelial Cells. Retinal endothelial cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 5, 25 and 50 mM Glucose, incubated for 24, 36 and 48 hours in humidified 5 % CO$_2$ incubator at 37$^{\circ}C$. Human Retinal Endothelial Cell Line (HREC) were characterized for morphology with different treatment by phase contrast microscopic analysis. Number of dead and viable cells was counted by trypan blue exclusion and supported by MTT assay. The intracellular Hydrogen peroxide (H$_2$O$_2$), a Reactive Oxygen Species (ROS) generation in high glucose conditions was assessed by FOX II assay and apoptosis by caspase-3 assay. The high glucose treated cells undergoing DNA fragmentation was witnessed by Agarose gel electrophoresis. We found that the cells incubated with 25 and 50 mM glucose containing medium for 48 hours altered the morphology of the cell, induced apoptosis and DNA fragmentation. The dead cell number were high in 25 and 50 mM when compared to the cells incubated with 5 mM glucose for 24, 36, and 48 hours. Also, the H$_2$O$_2$ levels and the activity of caspase-3 were increased in high glucose treated cells. Conclusions/interpretation: Our results demonstrated that elevated glucose induces apoptosis in cultured HREC. The hyperglycemia-induced increase in apoptosis may be dependent on caspase activation. The association between ROS generation and caspase-3 activation on high glucose treated cells is yet to be investigated.

Comparison of Stress Response in Diallel Crossed Korean Domestic Chicken Breeds (토종 종계를 이용한 이면 교배조합 계통 간 스트레스 반응정도 비교 분석)

  • Cho, Eun Jung;Park, Ji Ae;Choi, Eun Sik;Sohn, Sea Hwan
    • Korean Journal of Poultry Science
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    • v.43 no.2
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    • pp.77-88
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    • 2016
  • To establish a new synthetic Korean meat chicken breed, we tested $5{\times}5$ diallel cross mating experiment with domestic chicken breeds. Comparing stress responses among diallel crossed chicken breeds, we analyzed telomere length, DNA damage and expressions of heat shock protein genes (HSPs) as the markers of the stress response. The telomere length was measured by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes. The expression levels of HSP-70, $HSP-90{\alpha}$ and $HSP-90{\beta}$ genes were analyzed by quantitative real-time polymerase chain reaction in lymphocytes. The DNA damage rate of lymphocytes was quantified by the comet assay known as the single cell gel electrophoresis. In results, there were significant differences in the values of the stress markers such as telomere length, HSPs and DNA damage rate, and also were significant differences in viabilities and body weights among the $5{\times}5$ diallel crossed chicken breeds. The telomere shortening rate, expression values of HSPs and DNA damage rate were significant low in W and Y crossed chickens compare to the others, but GG pure breed showed the highest values in the 25 crossed chickens. Estimating correlation coefficient, the survival rate positively correlated to telomere length, but negatively correlated to the expression levels of HSP-70, $HSP-90{\alpha}$, $HSP-90{\beta}$ genes and to the value of % DNA in tail as DNA damage rate. The expression levels of HSP-70, $HSP-90{\alpha}$ and $HSP-90{\beta}$ genes of dead chickens had significantly higher than those of survival chickens. According to the results on the stress marker analysis, it would be considered that the crossed breeds had more stress resistant than the pure breeds, and the crossed chickens with a light strain such as W or Y were relatively resistant to stress, but the crossed chickens with a heavy strain such as G, H, F were susceptible to stress.

Changes of the blood chemistry, lipid and protein components in blood and liver tissue according to the time lapsed of the rat after oral administration of caffeine (Rat에 caffeine 경구투여후 시간경과별로 혈액과 간조직에서 혈액화학성분, 지질 및 단백질 구성성분의 변화)

  • Do, Jae-cheul;Huh, Rhin-sou
    • Korean Journal of Veterinary Research
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    • v.36 no.4
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    • pp.795-807
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    • 1996
  • This study was conducted to identify the effects of caffeine 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. Acute test were conducted to determine those effects. The acute test was conducted by dividing rats into 7 groups according to the time lapsed after a single oral administration of 100mg/kg caffeine(that is control, 2hrs, 4hrs, 8hrs, 24hrs, 48hrs and 72hrs lapsed group). 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, malondialdehyde(MDA) and the patterns of the SDS-PAGE(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) 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. The concentrations of serum glucose were significantly higher(p<0.01) between 4(143.0mg/dl) and 8hrs(138.0mg/dl) in comparison to that of the control(101.1mg/dl) after a single oral administration of caffeine(100mg/kg). While on the other, there were no significant differences in the concentrations of urea nitrogen, uric acid, creatinine, total protein, albumin and albumin/globulin(A/G) ratio in comparison to those of the control. 2. The concentrations of total cholesterol and HDL-cholesterol in serum were significantly higher(p<0.01) between 4(77.4mg/dl, total cholesterol) and 8hrs(64.7mg/dl, HDL-cholesterol) in comparison to those of the control(62.8, 46.7mg/dl) after a single oral administration of caffeine(100mg/kg). On the other hand, the concentrations of triglyceride in serum were significantly lower(p<0.01) after 8hrs(38.8mg/dl) in comparison to that of the control(66.5mg/dl). 3. The activities of AST in serum was significantly higher(p<0.05) from 2hrs(149U/L) to 8hrs(178U/L) in comparison to the control(112U/L) after a single oral administration of caffeine(100mg/kg). The activities of ALT in serum were significantly higher(p<0.01) at 4(45.5U/L), 24(49.3U/L), 48(46.8U/L) and 72 hrs(42.3U/L) in comparison to that of the control(39.7U/L) after a single oral administration of caffeine(100mg/kg). On the other hand, there were no significant differences in the activities of ALP in comparison to that of the control. 4. The concentrations of free fatty acid in serum were significantly higher(p<0.01) at 8hrs(65.0mg/dl) in comparison to that of the control(37.6mg/dl) after a single oral administration of caffeine(100mg/kg). However, there were no significant differences in the concentrations of carbonyl group and malondialdehyde within serum, and liver homogenate, mitochondrial and microsomal fractions in comparison to that of the control. 5. The patterns of SDS-PAGE in serum, mitochondrial and microsomal fraction of the liver showed no significant differences.

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Catalases in Acinetobacter sp. Strain JC1 DSM 3803 Growing on Glucose (포도당을 이용하여 성장하는 Acinetobacter sp. Strain JC1 DSM 3809에 존재하는 Catalase)

  • Shin, Kyoung-Ju;Ro, Young-Tae;Kim, Young-Min
    • Korean Journal of Microbiology
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    • v.32 no.2
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    • pp.155-162
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    • 1994
  • Cells of Acinetobacter sp. strain JC1 DSM 3803, an aerobic monoxide-oxidizing bacterium, growing on glucose exhibited high catalase activity at the mid-exponential growth phase. The enzyme activity decreased gradually after then until the early stationary phase, increased again at the mid-stationary phase, and then decreased again thereafter. Cells growing on glucose was found to contain three kinds of catalses. Cat1, Cat2 and Cat3. The activities of Cat1 and Cat3 did change significantly during growth, but that of Cat2 exhibited significant variation. Cat3 was found to present only in cells growing on glucose, but not in cells growing on carbon monoxide of methanol. The activities of call and Cat3 in cell-free extracts were stable upon treatment with ethanol and chloroform, but decreased to some extent when the enzymewere treated with 2mM $H_2O_2$ and/or 3-amino-1,2,4-triazole (AT). Cat2 was found to be extremely sensitive to the ethanol-chloroform and $H_2O_2$ treatments, but was insensitive to the AT treatment. Cat1 exhibited enzyme activity after incubation for 1 min at 80$^{\circ}C$. Cat2 and Cat3 did not show enzyme activity after incubation for 1 min at 60$^{\circ}C$ and 70$^{\circ}C$, respectively. Cat2 was found to have peroxidase activity. Cat3 was purified to homogenity in seven steps. The molecular weight of the native enzyme was estimated to be 150,000. Sodium dodecyl sulfate-gel electrophoresis revealed two identical subunits of molecular weight 65,000. The enzyme was found to show two $K_m$ values of 39 mM and 58mM. The optimal pH for the enzyme activity was 7.0, but the activities at pH 6.0, 8.0, and 9.0, were found to be comparable to that at the optimal pH. The optimal temperature for the enzyme activity was found to be 40$^{\circ}C$. The enzyme also exhibited strong activity at 20$^{\circ}C$, 30$^{\circ}C$, and 50$^{\circ}C$. The purified enzyme was not affected by the ethanol-chloroform treatment. The enzyme, howerver, showed less than 10% of the original activity when it was treated with 12 mN AT, 0.1 mM $NaN_3$ of 1mM KCN.

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Efficient Remediation of Petroleum Hydrocarbon-Contaminated Soils through Sequential Fenton Oxidation and Biological Treatment Processes (펜톤산화 및 생물학적 연속처리를 통한 유류오염토양의 효율적 처리)

  • Bae, Jae-Sang;Kim, Jong-Hyang;Choi, Jung-Hye;Ekpeghere, Kalu I.;Kim, Soo-Gon;Koh, Sung-Cheol
    • Korean Journal of Microbiology
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    • v.47 no.4
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    • pp.356-363
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    • 2011
  • The accidental releases of total petroleum hydrocarbons (TPH) due to oil spills frequently ended up with soil and ground water pollution. TPH may be degraded through physicochemical and biological processes in the environment but with relatively slow rates. In this study an attempt has been made to develop an integrated chemical and biological treatment technology in order to establish an efficient and environment-friendly restoration technology for the TPH contaminated soils. A Fenton-like reaction was employed as a preceding chemical treatment process and a bioaugmentation process utilizing a diesel fuel degrader consortium was subsequently applied as a biological treatment process. An efficient chemical removal of TPH from soils occurred when the surfactant OP-10S (0.05%) and oxidants ($FeSO_4$ 4%, and $H_2O_2$ 5%) were used. Bioaugmentation of the degrader consortium into the soil slurry led to an increase in their population density at least two orders of magnitude, indicating a good survival of the degradative populations in the contaminated soils ($10^8-10^9$ CFU/g slurry). TPH removal efficiencies for the Fenton-treated soils increased by at least 57% when the soils were subjected to bioaugmentation of the degradative consortium. However, relatively lower TPH treatment efficiencies (79-83%) have been observed in the soils treated with Fenton and the degraders as opposed to the control (95%) that was left with no treatment. This appeared to be due to the presence of free radicals and other oxidative products generated during the Fenton treatment which might inhibit their degradation activity. The findings in this study will contribute to development of efficient bioremediation treatment technologies for TPH-contaminated soils and sediments in the environment.

Comparisons of Meat Quality Characteristics between Castration and Non-castration from Dairy Goats (거세 및 비거세 유산양고기의 품질특성 비교)

  • Kang, Geunho;Cho, Soohyun;Seong, Pilnam;Kang, Sunmun;Park, Kyoungmi;Park, Beomyoung;Kim, Donghun
    • Food Science of Animal Resources
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    • v.33 no.1
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    • pp.119-124
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    • 2013
  • This study was conducted to investigate meat quality and sensory characteristics between castrated and non-castrated dairy goats. Dairy goat of Saanen breeds was slaughtered at an age of 6 mon. Then, characteristics of dairy goat meat were analyzed to chemical compositions, collagen content, pH, meat color, cooking loss, water-holding capacity, shear force, protein solubility, and myofibrillar protein fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Also, odor from dairy goat meats was analyzed by sensory evaluation and volatile substances by gas chromatography-mass spectrometry (GC-MS). As a result, the chemical compositions and physicochemical characteristics were not significantly different between castrated and non-castrated dairy goats meat. Also, there is no difference protein solubility (sarcoplasmic, myofibrillar and total protein) and protein fraction by SDS-PAGE. Sensory evaluation results in odour scores are highly (p<0.05) non-castration dairy goat meat better than castration. As a result, overall palatability was higher (p<0.05) in castrated goat meat when compared with non-castrated one. The indole and octadecanoic acid by GC-MS based on sensory evaluation results were only detected in non-castrated dairy goat meat. Therefore, distribution for goat meats castrated compared to non-castrated dairy goat meat is expected to be able to get a good response to the Korean consumer.

Isolation, Purification and Some Properties of Polyphenol Oxidase from Pear (배과실(果實)의 Polyphenol Oxidase의 분리(分離) 정제(精製) 및 그 특성(特性))

  • Kang, Yoon Han;Sohn, Tae Hwa;Choi, Jong Uck
    • 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.

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