버섯 중 Aminotransferase 의 동질효소 분리정제 및 그 특성에 관한 연구(II) -표고버섯(Lentinus edodes)중의 Branched Chain Amino Acid Aminotransferase(Enzyme II)의 정제 및 특성-

민태진,배강규,Min, Tae-Jin,Bae, Kang-Gyu 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.6

Enzyme I and II of branched chain amino acid aminotransferase (BCAT) in Lentinus edodes (Berk.) Sing was purified by ammonium sulfate fractionation, DEAF-cellulose column chromatography and sephadex G-150 gel filtration. We have been studied molecular weight, N-terminal amino acid, composition of amino acids, optimum pH, optimum temperature, $K_m$ value and effect of organic compounds and metal ions about enzyme II, to identify isozyme patterns between enzyme I and II. Apparent molecular weight of enzyme II was estimated 72,000 dalton, N-terminal amino acid was determined glycine. The enzyme II was existed as negatively charged amino acids rather than positively charged ones by more than 3.67%. Optimum pH and temperature were found to be 8.2 and $38^{\circ}C$, respectively. It was stable for 30 min under $20^{\circ}C$. This enzyme showed the activity toward L-leucine, as a substrate. It was inhibited by both organic compounds of hydroxylamine, N-ethylmaleimide and p-chlromercuribenzoate and metal ions of $Cu^{2+}$, $Hg^{2+}$ and $Fe^{2+}$. The $K_m$ values were determined to be 3.39 mM for L-leucine as a amino group donor, 0.28 mM for $\alpha$-ketoglutarate as a amino group acceptor. Although their molecular weight, N-terminal amino acid, composition of amino acids, optimum pH and temperature and $K_m$ values had been different between enzyme I and II, but they showed same substrate specificity for L-leucine as a substrate. 표고버섯, Lentinus edodes (Berk.) Sing 중의 가지달린 아미노산 aminotransferase(BCAT)의 enzyme I과 II를 황산암모늄 분별침전, DEAE-cellulose column 크로마토그래피 및 Sephadex G-150 겔 여과하여 각각 분리정제한 후, enzyme I과 II의 동질효소 여부를 구명하기 위하여 enzyme II의 분자량, N-말단, 아미노산 조성, 최적 pH, 최적 온도, $K_m$값, 유기물 및 금속이온 효과 등을 실험하였다. 이 효소의 겉보기 분자량은 72,000 dalton 이었고, N-말단 아미노산은 L-leucine이었으며, 음성잔기를 가진 아미노산이 양성잔기를 가진 것보다 3.67% 더 많이 존재하였다. 최적 pH 및 최적 온도는 각각 8.2, $38^{\circ}C$ 였으며, $20^{\circ}C$이하에서 30분 동안 안정하였다. 이 효소는 기질인 가지달린 아미노산 중 L-leucine에 대해서만 활성을 보였으며, hydroxylamine, N-ethylmaleimide 및 p-chloromercuribenzoate 등의 유기물과 $Cu^{2+}$, $Hg^{2+}$ 및 $Fe^{3+}$ 등의 금속이온에 의하여 활성이 억제 되었다. 아미노기 공여체인 L-leucine과 아미노기 수용체인 $\alpha$-ketoglutarate에 대하여 활성도 측정시의 조건하에서 측정된 $K_m$값은 각각 3.39 및 0.28 mM이었다. 이로써 enzyme II와 I은 분자량, N-말단 아미노산, 아미노산 조성, 최적 pH 및 최적 온도 그리고 $K_m$값 등은 서로 다르지만, L-leucine 기질에 대하여 같은 기질 특이성을 보이는 효소였다.

버섯의 Adenosinetriphosphatase ( ATPase ) 에 관한 연구 (2) - 표고버섯 ( Lentinus edodes ) 중 정제 F1-ATPase 의 금속이온 및 음이온 효과 -

민태진,박혜련 ( Tae Jin Min,Hye Lyoun Park ) 한국균학회 1991 韓國菌學會誌 Vol.19 No.3

누에(Bombyx mori L)에 요소 및 Eleutherococcus Senticosus Maxim 첨식효과에 관한 연구

민태진,Min, Tae-Jin 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.2

누에에 요소 1,600 ppm(U) 및 가시오가피 메탄올 추출물 1,200 ppm(E) 수용액을 각각 뽕잎에 처리하여 첨식시켰다. Cellulose acetate(CA) 및 disc-polyacrylamide gel(PAG) 전기영동으로 5령기말 암컷누에의 체내단백질의 영통상의 변화 그리고 가스-크로 마토그라파에 의하여 체내지방산의 함량변화를 대조군(C)과 비교하였다. CA전기영동은 C군 및 U군에 서는 albumin이 주 pattern이었으나 E군은 ${\alpha}$-globulin이었다. disc-PAG 전기영동 결과 C군와 E군은 동일하였으나 U군의 pattern III은 a,b 두개로 분리되었다. Sephadex G-150으로 크로마토그라피한 첫째번 분획들을 disc-PAG 전기영동한 결과 다같이 2째번 pattern이 urease이었다. 또한 3째번 분획들을 정제하여 SDS-PAG 전기영동에 의한 걷보기 분자량은 다같이 46,000 및 26,000이었으나, U군에서 57,000인pattern 1개를 더 관찰하였다. 체내지방산은 6종류로, 미리스트산, 팔미트산, 스테아르산, 올레산, 리놀레산 및 리놀렌산 이었으나 C군에 비하여 E군 U군 순으로 크게 증가하였다. 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 ${\alpha}$-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 division 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 acids in 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.

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

민태진,이강협 ( Tae Jin Min,Kang Hyeob Lee ) 한국균학회 1991 韓國菌學會誌 Vol.19 No.1

Purification and Properties of Carboxyl Proteinase from the Poria cocos (SchW.) Wolf (I)

민태진,정광식,김재웅,Min, Tae-Jin,Chung, Kwang-Sik,Kim, Jae-Woong 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.3

복령[Poria cocos (Schw.) Wolf.]중의 carboxyl proteinase을 황산 암모늄 65% 포화용액, DEAE-cellulose 및 Sephadex G-75을 이용한 gel-filtration 방법으로 분리 정제하여 그 성질을 조사하였다. 이 버섯중에서 활성분획분 I과 II을 얻었으며, 활성분획분 II에 대하여 bovine hemoglobin을 기질로 사용하여 얻은 carboxyl proteinase의 활성도의 최적 pH는 2.0, 최적온도는 $70^{\circ}C$, pH 안정성은 1.2~2.5 및 열안정성은 $25^{\circ}C{\sim}75^{\circ}C$였다. 이 효소의 subunit는 1개였고, 18종의 아미노산으로 구성된 단백질이였으며, 겉보기 분자량은 25,500(PAGE)~23,000 (HPLC)였다. 이 효소는 carbobenzoxy-L-glutamyl-L-tyrosine (CBZ-glu-tyr.)의 glutaml-L-tyrosine간의 부위를 절단하는 특이성을 가졌었다. CBZ-glu-tyr.을 사용하여 측정한 Km 값은 0.29 mM 이였다. The enzymic properties of carboxyl proteinase isolated from the Poria cacos (Schw.) Wolf. were investigated. Two kinds of active fraction I and II were isolated from this mushroom. The active fraction II showed carboxyl proteinase activity to bovine hemoglobin substrate, and its optimum conditions were the followings; temperature, $70^{\circ}C$, pH stability, 1.2~2.5 and thermal stability, $25^{\circ}C{\sim}75^{\circ}C$. The enzyme appeared to be one subunit, and consisted of 18 kinds of amino acids. The apparent molecular weight were 25,500 (PAGE) and 23,000(HPLC), respectively. The enzyme appeared to hydrolyze peptide bond between glutaml-L-tyrosine. The Km value of this enzyme was 0.29mM when carbobenzoxy-L-tyrosine were used as a subtrate.

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.

버섯중 철이온에 활성화된 광감응성 Mitochondrial ATPase 에 관한 연구

민태진,이미애,박상신 ( Tae Jin Min,Mi Ae Lee,Sang Shin Park ) 한국균학회 1993 韓國菌學會誌 Vol.21 No.3

친화성 고분자 유도체의 합성 및 단백질의 분리정제에 관한 연구 - Benzoyl - AH - Sepharose 4B 의 합성 및 흰느타리버섯중 단백질의 분리정제 -

민태진,장흥배,최원기 ( Tae Jin Min,Hung Bae Chang,Won Ki Choi ) 한국균학회 1988 韓國菌學會誌 Vol.16 No.3

누에 ( Bombyx mori L . ) 의 요소첨식 효과에 관한 연구

민태진,김택영 ( Tae Jin Min,Taik Yung Kim ) 생화학분자생물학회 1978 BMB Reports Vol.11 No.3

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 108×107 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, U₄ 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 U₄ 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 U₄ group compared to six in the control group.

흰느타리버섯 ( Pleurotsu cornucopiae ( pers ) Rolland ) 중의 Protease 의 분리정제 및 그 성질에 관한 연구 ( 1 )

민태진,홍성일,김재웅 ( Tae Jin Min,Sung Il Hong,Jae Woong Kim ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.1

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℃, 3.0∼4.0 and 20∼40℃, 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.