Kinetic Mechanism and End-Product Regulation of Deoxyguanosine kinase from Beef Liver Mitochondria

Park,Inshik,Ives,David H. 東亞大學校附設遺傳工學硏究所 1996 遺傳工學硏究 Vol.- No.3

Initial-rate kinetic measurements with the affinity purified 2-deoxyguanosine (dGuo) kinase from beef liver mitochondria yielded reciprocal plots which converged on the abscissa, with either dGuo or ATP as the varied substrate. The limiting Km for dGuo was 4.7 μM, and that for ATP was 780 μM. One product, dGMP, was competitive with both substrates, while the other, ADP, was competitive with ATP and non-competitive with dGuo. Qualitatively identical results were obtained with an alternative substrate, dTTP, and with alternative product inhibitors, dIMP and dTDP. These results are consistent with a random BiBi kinetic mechanism, judging from the formation of a dead-end complex of the enzyme, dGuo and ADP. dGTP competes very strongly with ATP (Ki=0.03μM), but is non-competitive towards dGuo. The more weakly-bound dGDP is competitive with both substrates.

Inhibition of Korean Radish Peroxidase by Sodium Azide

Inshik Park 한국식품과학회 1994 Food Science and Biotechnology Vol.3 No.1

Protein Engineering of Deoxynucleoside Kinase from Lactobacillus acidophilus : Effect of Site - Directed Mutagenesis on Microbial Growth

Inshik Park,Eun-Ae Kim,Keuk-Seung Bang,Seok-Hwan Kim,Gi-Nahm Kim Min-Kyung Lee,Ji-Oeun Kil 한국식품영양과학회 2001 Preventive Nutrition and Food Science Vol.6 No.1

Deoxynucleoside kinases exist as heterodimeric pairs specific for deoxyadenosine/deoxyguanosine kinase (dAK/dGK) and deoxyadenosine/deoxycytidine kinase (dAK/dCK). The aspartic acid-84 in dGK was mutated to alanine, asparagine and glutamic acid by site-directed mutagenesis. The mutation resulted in a drastic decrease in dGK activity compared to the unmodified cloned enzyme while it increased production of dAK activity. The mutated dak/dgk genes, which synthesize tandem deoxyadenosine/deoxyguanosine kinase, were inserted back to the Lactobacillus acidophilus and Lacto-coccus lactis by electroporation to determine the effect of site-directed mutation of the enzymes on the microbial growth. However, no significant change was observed in cell growth and lactic acid production between wild type and mutant lactic acid bacteria.

Antioxidant Activities of Fractions from Sedum sarmentosum

Inshik Park,Choon Young Kim,Min-Young Lee 한국식품영양과학회 2006 Preventive Nutrition and Food Science Vol.11 No.2

This study was conducted to evaluate the antioxidant activity of each fraction from Sedum sarmentosum. Antioxidant activity of each fraction was measured using the DPPH radical assay, the ferric thiocyanate (FTC) method, and thiobarbituric acid (TBA) method. The antioxidant activities were then compared with that of BHT (synthetic antioxidant). The ethyl acetate and butanol fractions were found to have significant DPPH radical scavenging activity, with scavenging potencies showing 90.61% and 87.02%, respectively. Total phenolic compound contents, determined according to the Folin-Denis method, were found to be in the order of ethyl acetate>butanol>ethanol>chloroform>aqueous fraction. From the results, we have been able to establish a positive correlation between the antioxidant activity and the total phenolic compound content of the sample. The antioxidant activity in a linoleic acid system was measured using the ferric thiocyanate (FTC) method and thiobarbituric acid (TBA) method. The ethyl acetate fraction had the highest antioxidant activity among the tested fractions. On the basis of these results, the ethyl acetate fraction provided equivalent or higher antioxidant activity as compared to BHT. These results suggest that Sedum sarmentosum is a potentially useful antioxidant for foods, cosmetics, and medicine.

The Novel Assay Method for Thrombin by Weighing Fibrin Clot

Park, Inshik,Kim, Gi-Nahm Korean Society of Life Science 2004 생명과학회지 Vol.14 No.3

본 연구는 피브린의 무게를 측정하는 방법에 의하여 간단하고 단순하게 트롬빈활성을 측정하는 방법을 확립하기 위하여 수행되었다. 이 새로운 트롬빈측정 법은 트롬빈이 기질인 피브리노겐과 반응 후 생성된 피브린의 무게를 측정한다. 피브린의 생성은 사용한 트롬빈의 효소활성, 반응온도, 반응시간 및 원심분리의 시간에 의존하였다. 피브린의 생성은 37$^{\circ}C$에서 트롬빈활성이 1.0 unit/ml 이내, 4.0mg/ml의 피브리노겐 농도 및 반응시간이 5분 이내에는 직선적으로 증가하였다. 그리고 생성된 피브린은 3,000$\times$g에서 5분간 원심분리의 경우에 안정하였다. 이 새로운 피브린 측정 법은 트롬빈을 저해하는 천연물질을 탐색하는데 유행하게 사용될 것으로 기대된다. This study was performed to establish a simple and rapid method for measuring thrombin activity based on weight of fibrin clot formed. The new method was based on the weight measurement of fibrin clot after enzymatic reaction of thrombin with fibrinogen. The fibrin formation depended upon the activities of thrombin used, temperature, incubation time, and centrifugation time. The fibrin formation was increased proportionally up to 1.0 unit/ml of thrombin activity, 4.0mg/ml of fibrinogen concentration, and 5 min of incubation time at 37$^{\circ}C$. The fibrin clot formed was stable by centrifugation at 3,000$\times$g for 5min. This simple assay based on weight of fibrin after centrifugation would be useful for identifying natural food anticoagulants by inhibiting thrombin.

Protein Engineering of Deoxynucleoside Kinase from Lactobacillus acidophilus: Effect of Site-Directed Mutagenesis on Microbial Growth

Park, Inshik,Kim, Eun-Ae,Bang, Keuk-Seung,Kim, Seok-Hwan,Kim, Gi-Nahm,Lee, Min-Kyung,Kil, Ji-Oeun The Korean Society of Food Science and Nutrition 2001 Preventive Nutrition and Food Science Vol.6 No.1

Deoxynucleoside kinases exist as heterodimeric pairs specific for deoxyadenosine/deoxyguanosine kinase (dAK/dGK) and deoxyadenosine/deoxycytidine kinase (dAK/dCK). The aspartic acid-84 in dGK was mutated to alanine, asparagine and glutamic acid by site-directed mutagenesis. The mutation resulted in a drastic decease in dGK activity compared to the unmodified cloned enzyme while it increased production of dAK activity. The mutated dak/dgk genes, which synthesize tandem deoxyadenosine/deoxyguanosine kinase, were inserted back to the Lactobacillus acidophilus and Lactococcus lactis by electroporation to determine the effect of site-directed mutation of he enzymes on the microbial growth. However, no significant change was observed in cell growth and lactic acid production between wild type and mutant lactic acid bacteria.

Inhibition of Korean Radish Peroxidase by Sodium Azide

Park,Inshik 東亞大學校附設遺傳工學硏究所 1995 遺傳工學硏究 Vol.- No.2

Sodium azide inhibited peroxidase from Korean radish strongly at acidic pH ranges. The inhibitory effect of sodium azide was drastically increased by addition of hydrogen peroxide. The rapid decrease of the enzyme activity in the presence of both sodium azide and hydrogen peroxide seems due to irreversible inactivation of the enzyme by them. However, the enzyme seems to be inhibited reversibly in the presence of sodium azide only.

Efficient Extraction of Enzyme Localized in Cell Wall of Yeast

Park, Inshik,Kill, Ji-Oeun,Kim, Gi-Nahm 東亞大學校附設遺傳工學硏究所 1997 遺傳工學硏究 Vol.- No.4

Invertase was effectively extracted from Candida utilis cells using various reducing agents such as 2-mercaptoethanol, dithiohreitol, or cysteine. The extraction of the enzyme depended upon the kind and concentration of reducing agents used, pH, temperature, and time of incubation. The enzyme can be optimally extracted by incubating the cells with 10 mM 2-mercaptoethanol at 50℃ after adjusting the pH of either cell precipitate or culture broth to 7.0. The enzyme can be extracted from either cell precipitate or culture broth directly without preparative cell harvesting.

Optimal Culture Condition for Production of Acidic Glucoamylase from Aspergillus sp.

Park,Inshik,Chung,Youngho 東亞大學校附設基礎科學硏究所 1987 基礎科學硏究論文集 Vol.4 No.1

토양으로부터 분리된 내산성 extracellular glucoamylase를 생성하는 Aspergillus 곰팡이의 최대효소생산 조건을 검토하였다. 효소 생산에 있어서 탄소원으로서는 soluble starch가 5%수준에서 가장 우수했으며 질소원으로서는 yeast extract가 1% 수준에서 가장 적합하였다. 그리고, 최대효소생성을 위해서는 배양액의 pH는 6.0, 배양온도는 28℃가 최적임을 확인하였다. 금속이온중 ??을 첨가시 효소생성이 촉진됐으며 ??는 효소생성 및 곰팡이의 생육을 억제하였다. 고체배양의 경우에는 밀기울이 가장 효과가 좋았으며 배양온도는 30℃에서 가장 효과가 높았다. Production of acidic glucoamylase by Aspergillus sp. isolated from soil was examined under various culture condition. Initial pH adjustment of culture medium in pH 6.0 was found to be necessary for optimal production of the enzyme. The optimal cultivation temperature was found to be 28℃ for liquid culture, and 30℃ for solid(wheat bran) culture. Among tested carbon and nitrogen sources, soluble starch and yeast extract were found to be best for the enzyme production. The optimal soluble starch and yeast extract concentrations for the enzyme prpduction were 5% and 1%, respectively. Among added metal ions tested, ?? showed increasing effect for the enzyme production, while ?? not only inhibited the growth of the organism but also decreased the production of the of the enzyme effectively.

Activation of Cabbage Peroxidase by Imidazole at Alkaline pH

Park, Kui-Sun,Lee, Mi-Kwang,Kim, Seok-Hwan,Park, Inshik,Kim, Kyung-Ja 東亞大學校附設遺傳工學硏究所 1997 遺傳工學硏究 Vol.- No.4

Peroxidase(EC 1.11.1.7) from cabbage could be activated by adding various nitrogenous compounds. Among nitrogenous compounds tested, imidazole was the best activator of the enzyme. Imidazole activated the enzyme at alkaline pH, while it had no effect at acidic pH. The optimum concentration of imidazole to stimulate the enzyme was identified as 20 mM. Imidazole seemed to activate the enzyme with anilino substrate such as o-dianisidine, while it had no effect with phenolic substrates such as guaiacol and 4-aminoantipyrine phenol.