실험실탐방 : 민태진 교수 실험실

민태진 한국생화학분자생물학회 (구 한국생화학회) 1990 생화학분자생물학회 소식 Vol.10 No.3

흰느타리버섯(Pleurotsu cornucopiae(pers) Rolland)중의 Protease의 분리정제 및 그 성질에 관한 연구(I)

민태진,홍성일,김재웅,Min, Tae-Jin,Hong, Sung-Il,Kim, Jae-Woong 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.1

흰느타리버섯(Pleurotus cormkopiae(pers.) Rolland)중의 protease를 DEAE-sephadex A-50, CM-cellulose, DEAE-cellulose 및 Sephadex G-75를 사용하여 분리정제하고 그 성질을 조사하였다. 이 버섯중에는 bovine serum albumin을 기질로 하였을 때 2종류의 활성분획을 함유하고 있었고, protease(분획 I)의 최적 pH는 4.0, 최적온도는 $50^{\circ}C$, pH 안정도는 pH 3.0~4.0 그리고 열안정도는 $40^{\circ}C$ 이하에서 안정하였다. 또한$Ca^{++}$, $Cu^{++}$, $Zn^{++}$, $Fe^{++}$ 및 $Mg^{++}$ 이온은 이 효소의 활성도를 저해하였으나, $Co^{++}$ 이온은 효소의 활성을 증가시켰다. 이 효소의 subunit는 1개 였고, 걷보기 분자량은 56,000 daltons이었으며 아미노산 조성은 17종이었다. The enzyme properties of protease of the Pleurotus Cornucopiae (Pers.) Rolland were investigated by purification with gel filteration using DEAE-sephadex A-50, CM-cellulose, DEAE-cellulose and Sephadex G-75. Two kinds of active fractions were isolated from this mushroom study. The active fraction showed protease activity for the bovine serum albumin substrate, and its optimum pH, optimum temperature, pH stability and thermal stability were 4.0, $50^{\circ}C$, 3.0~4.0 and $20{\sim}40^{\circ}C$, respectively. The activity of the enzyme was inhibited by $Ca^{++}$, $Cu^{++}$, $Zn^{++}$, $Fe^{++}$ and $Mg^{++}$, but increased with $Co^{++}$. The enzyme had one subunits composed of 17 amino acids. The apparent molecular weight was about 56,000 daltons.

누에(Bombyx mori L.)의 요소첨식 효과에 관한 연구

민태진,김택영,Min, Tae-Jin,Kim, Taik-Yung 생화학분자생물학회 1978 한국생화학회지 Vol.11 No.3

누에(수원 $108{\times}107$호 교잡종)에 3령기에서 5령기까지 요소를 농도별(1,000ppm~2,000ppm)로 첨식시켜 성장도, 고치량의 변화, 체내 Urease의 활성도, 핵산, 단백질의 함량 변화 및 Acrylamide gel올 이용한 disc electrophoresis를 통하여 단백질의 이동상의 변화를 관찰하였다. 요소 1600ppm($U_4$) 처리구에서, 성장도 및 고치량의 증가율은 각각17.43%, 20.11%로 최고치였다. Urease의 활성도는 3령기에서 5령기로 갈수록 크게 감소하였고, 요소의 농도 증가에 따라 증가하나 $U_4$ 이상에서는 감소하였다. 그러나 요소처리구는 대조구에 비하여 감소하였다. 핵산량은 3령기에서 5령기로 갈수록 크게 감소하고, 요소처리구는 대조구에 비하여 감소하지만 요소 농도 증가에 따라 증가하였다. 5령기 끝날, 누에의 체내 단백질의 전기이동상은 대조구(C)에서 6개, $U_4$구에서는 5개의 이동상을 확인하였다. The change of the growth rate, the weight of cocoon, and the change of Urease activity, nucleic acid, protein contents, and the changes in disc electrophoretic patterns of the protein in the silkworm (Suwon No $108{\times}107$ confused species) from the 3rd instar stage to the 5th instar stage by supplemental feeding of different concentrations of Urea has been studied. On the experiment group, $U_4$ which was treated with 1,600ppm Urea, the rate of growth increased to 17.43% and the rate of cocoon's weight increased 20.11% more than the control groups. The activity of Urease was increased with the increasing of the concentration of Urea treatment, but not more than the control groups. Beyond $U_4$ activity of Urease was decreased gradually with the increasing of the concentration of Urea. The contents of nucleic acid were decreased sharply at first in the Urea treatments but gradually increased according to the Urea concentration. In the 5th instar stage, disc electrophoretic patterns of protein in. the silkworms were found to be five in the $U_4$ group compared to six in the control group.

흰느타리버섯(Pleurotus cornucopiae (per.) Rolland)중의 Protease의 분리정제 및 성질에 과한 연구 (II)

민태진,이수용,김재웅,Min, Tae-Jin,Lee, Soo-Young,Kim, Jae-Woong 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.2

흰느타리버섯(Pleurotus cornucopiae (Per.) Rolland)의 protease를 분리 정제하여 그 성질을 조사하였다. Hippuryl-L-phenylalanine 기질에 대한 이 효소의 $K_m$ 값은 0.45 mM 이었고, glycyl-L-phenylalanine의 펩티드 결합을 분해시키는 특이성을 보였다. L-Cysteine, L-Lysine, DAN, EPNP, EDTA, ${SO_3}^{-2}$, ${SO_4}^{-2}$, 및 ${HPO_4}^{-2}$은 효소활성을 저해하였고, ${NO_2}^{-1}$은 활성을 증가시켰다. L-Cysteine은 경쟁적 방해제로 작용하였고, 그것의 $K_i$값은 2.4 mM 이었다. $pK_a$값은 4.4였다. The enzymic properties of protease in the Pleurotus cornucopiae(Per.) Rolland were investigated. The $K_m$ value was 0.45 mM when hippuryl-L-phenylalanine was used as a substrate. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-phenylalanine. This enzyme was inhibited by L-lysine, diazoacetyl-DL-norleucine methyl ester(DAN), 1,2-epoxy-(p-nitrophenoxy) propane(EPNP) and EDTA, also was inhibited by anion as ${SO_3}^{-2}$, ${SO_4}^{-2}$ and ${HPO_4}^{-2}$ but increased with ${NO_2}^{-1}$ ion. The L-cysteine was found to be competitive inhibitor and its $K_i$ value was determined to be 2.4 mM by Dixon plot.

느타리버섯 중의 Light-Induced Mitochondrial ATPase에 관한 연구 : 유기물 효과 Effects of organic compounds

이호연,민태진 동국대학교 자연과학연구소 1991 자연과학연구 논문집 Vol.11 No.-

담자균 중의 미토콘드리아성 ATPase와 고등동물 세포중의 미토콘드리아성 ATPase의 유기물 효과를 비교 검토하기 위하여, 느타리버섯중의 미토콘드리아를 설탕밀도 비선형 기울기 원심분리법으로 분리 정제하였다. 유기물 중 미토콘드리아의 내막에 결합된 ATPase의 그 활성을 억제하는 물질로 알려진 N,N'-dicychohexylcarbodiimide(DCCD), 산화적 인산화 반응의 uncoupler로 알려진 carbonylcyanide m-chlorophenylhydrazone(CCCP), Fe 효소의 억제제이며 uncoupler로 알려진 sodiumazide, 그리고 산화적 인산화 및 인산기 전이의 억제제로 알려진 aurovertin B가 느타리버섯중의 미토콘드리아성 ATPase의 활성에 미치는 영향을 연구하였다. 이 효소의 활성은 0.25 mM DCCD, 0.02 mM sodium azide 및 eksqorwf mg당 1.5㎍의 aurovertin B에 각각 64%, 57%, 그리고 53% 감소하였다. 그러나 0.15μM CCCP에 의하여는 22%의 활성을 촉진시켰다. Mitochondriain Pleurotus ostreatus was isolated and purified by stepped sucrose density gradient centrifugation, in order to compare the effects of organic compound on the activity of mitochondrial ATPase in Basidiopmycota with that in mammalian cell. The effects of N,N'-dicychohexylcarbodiimide(DCCD) known as inhibitor of membrane bound ATPase, carbonylcyanide m-chlorophenylhydrazone(CCCP) known as uncoupler and inhibitor of oxidative phosphorylatioin, sodium azide known as inhibitor of Fe enzyme of uncoupler and aurovertin B known as preventing phosphoryl group transfer on the activity of mitochondrial ATPase were investigated. The activity of mitochondrial ATPase was inhibited by 64%, 57% and 53% in the presence of 0.25 mM DCCD, 0.02 mM sodium azide and 1.5 (㎍/mg of protein) aurovertin B, respectively. But it was stimulated by 22% in the presence of 0.15μM CCCP.

복령의 Carboxyl Proteinase 의 분리 정제 및 그 성질에 관한 연구 (2)

민태진,박상신,문순구 한국균학회 1986 韓國菌學會誌 Vol.14 No.2

The properties of carboxyl proteinase which was contained is Poria cocos (Schw.) Wolf were investigated by means of the purification with 0.65 ammonium sulfate saturation, DEAE-cellulose sad Sephadex G-75 gel filtration. This enzyme was found to hydrolyze only peptide bond between glutamyl-L-tyrosine of carbobenzoxy-L-glutamyl-L-tyrosine among the synthetic substrates of carbobenzoxy-L-glutamyl-L-tyrosine, hippuryl-L-phenylalanine and hippuryl-L-arginine. This enzyme was inhibited by Zn^(+2), Fe^(+2), Ca^(+2), CN^(-1), P₂O_7^(-1) ions, but stimulated by Hg^(+2) ion. Also, this enzyme was inhibited by organic compounds such as L-lysine, L-phenylalanine, hippuryl-L-phenylalaniae, diazoacetyl-DL-norleucine methyl ester (DAN) and 1.2-epoxy-3-(P-nitrophenoxy)propane(EPNP). In particular, the activity was inhibited by L-lysine till 20 minutes of preincubation time rapidly, and by DAN in the presence of Cu^(+2) ion more rapidly after 30 minutes than DAN in the absence of Cu^(+2) ion. L-Lysine was found to be a competitive inhibitor and its K_i value was determined to be 0.12 mmole by Dixon plot.

누에의 尿素添食 效果에 관한 硏究(Ⅰ)

閔泰鎭 東國大學校 1977 論文集 Vol.16 No.-

On this study I tried to find the change of the growth rate, the weight of cocoons and the change of Urease activity, RNA and DNA contents in the silkworm by the supplemental feeding of Urea. On the experiment group, U4 which was treated with 1000 ppm Urea, the rate of growth increased to 25.9% and the cocoon's weight increased 24.9% more than the control groups. The activity of Urease increased with the increasing of the concentration of Urea treatment, but not more than the control groups and on the 5th instar stage, their activity of Urease decreased again with their day age. The RNA contents increased gradually with the increasing of the concentration of urea treatments. The DNA contents was lower than the control group but with increasing of the Urea concentration, increased again gradually. Above those was almost same with the growth curve of the larvae but all those contents shown the decreasing with the material's day age.

무흡광 색소 생물의 감광수용체 개발 연구 (4) - 표고버섯 중의 광감응성 Mitochondrial ATP synthase 의 유기물 및 금속이온 유입효과 -

민태진,이완기,김재웅,민태익 ( Tae Jin Min,Wan Gie Lee,Jae Woong Kim,Tae Ick Mheen ) 한국균학회 1989 韓國菌學會誌 Vol.17 No.2

느타리버섯 (Pleurotus ostreatus) 중의 Light - induced mitochondrial ATP Synthase 의 효소적 특성에 관한 연구

민태진,이강협 한국균학회 1991 韓國菌學會誌 Vol.19 No.1

Mitochondria in Pleurotus ostreatus were isolated and purified by the stepped sucrose density gradient centrifugation, to investigate the effects of the light on the enzymatic activity of the mitochondrial ATP synthase. This enzyme, which was illuminated by the light ranging from 400 ㎚ to 700 ㎚, showed that the specific activity was stimulated at 490 ㎚ for 15 sec. Effects of organic compounds on the mitochondrial ATP synthase were also investigated at the optimum conditions; The activities of this enzyme were increased to 168 percent by the addition of 2,6-dichlorophenol indophenol(DCPIP), 224 percent by phenazine methosulfate(PMS), but inhibited 91 percent by oligomycin, 14 percent by 2-heptyl-4-hydroxyquinoline-N-oxide(HQNO) and 75 percent by 2,4-dinitrophenol (DNP), respectively. Effects of metal ions of the mitochondrial ATP synthase were investigated at the optimum conditions. The activities of the enzyme were inhibited 35 percent by Ca^(2+), 14 percent by Co^(2+) and 73 percent by Mn^(2+). For effects of anions, the activities of this enzyme were inhibited 80 percent by CN^-, 52 percent by SO₄^(2+), 28 percent by each of CO₃^(2+) and NO₃^-, respectively.

尿素添食에 따른 누에 體內蛋白質 및 脂肪酸에 關한 硏究

閔泰鎭,민태원 동국대학교 1982 論文集 Vol.21 No.-

Two groups of the silkworms feeding on mulberry leaves treated with solution of 1,600 ppm of urea (U) and 1,200 ppm of methanol extract of Eleutherococcus Senticosus maxim (E) respectively, in comparison with control group (C) were observed. On the end of the fifth instar stage of the female silkworm, significant differences were observed in the body fluid's protein patterns and contents of fatty acid of treatment groups compared with control group. The main patterns of CA-electrophoresis in the C group and U group were albumin and α-globulin in the E group. Pattern Ⅲ by disc-polyacrylamide gel (PAG) electrophoresis was double patterns in the U group and a single pattern in C and E group. In the three groups, second pattern by disc-PAG electrophoresis of the first divsion from sephadex G-150 gel fractionation was urease. The apparent molecular weights in the purified third division by SDS-PAG electrophoresis were, in the three groups, 46,000 and 26,000. However, another pattern with 57,000 was observed in the U group. Fatty acid in the silkworms by gas-chromatography contents myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. Their content were the greatest in the U group followed by E group and C group.