연어알에서 분리한 Carotenoprotein의 구조적 특성

김재웅,민태진,이태녕,Jae-Woong Kim,Tae-Jin Min,Tae-Young Lee 대한화학회 1988 대한화학회지 Vol.32 No.4

Carotenoprotein from Salmo Salar eggs was purified and characterized by CM-cellulose, 50% $(NH_4)_2SO_4$, DEAE-cellulose and sephadex G-75 column. The chromoprotein had a spectrum with ${\lambda}_{max}$ 409, 540 and 580nm in p-buffer (pH 7.0) at initial step. Molecular weights by sephadex G-200 gel filtration were 50, 200 and 26,000 daltons. SDS-PAGE analysis showed a structure with four identical subunits (12,500 daltons). Its sample retained a small amount of carbohydrates and lipids. Amino acids were analyzed, and mannose, galactose and glucosamine also were identified. Carotenoid extacted with acetone was found to be astaxanthin ester by partition test, epoxy test, iodine test, allylic test, reduction, acetylation, uv/vis, ir and nmr datas. Stearate (47.9%) and palmitate (21.4%) were predominant fatty acids in the astaxanthin ester. 연어알(Salmo Salar)에서 carotenoprotein을 CM-세룰로스, 50% 황산암모늄 침전, DEAE-세룰로스 및 세파덱스 G-75젤을 사용하여 충분히 정제하였다. 인산염 완충용액(pH 7.0)에서 자외선 흡수극대는 ${\lambda}_{max}$ 409, 540 그리고 580nm이었다. 세파덱스 G-200젤 여과법으로 측정된 분자량은 50, 200rhk 26,000달톤으로 heterogeneous한 두가지 형태였다. 전자는 적자색으로 SDS-PAG 전기영동 결과 분자량이 12,500달톤인 4개의 동일한 subunit로 구성되었고, 상당량의 지질과 만노오스, 갈락토스와 글루코사민을 함유하고 있음을 밝혔다. carotenoprotein의 색소를 추출하여 tlc로 정제한 다음 분배비 시험, 요오드 시험, 에폭시 그룹 시험, 알릴릭 시험, 환원 시험 그리고 아세틸화 시험등의 유기반응과 uv/vis, ir 및 nmr 데이타를 얻어 구조를 결정한 결과 carotenoid는 astaxanthin ester임을 확인하였으며 그 ester는 스테아르산(47.9%)과 팔미트산 (21.4%)이 대부분이었다.

사철느타리버섯 중 $G_{418}$-sensitive 미토콘드리아성 ATPase/ATP synthase의 특성

김재웅,김동희,이정복,이서구,민태진,Kim, Jae-Woong,Kim, Dong-Hee,Lee, Jung-Bock,Lee, Sur-Koo,Min, Tae-Jin 한국분석과학회 1992 분석과학 Vol.5 No.4

형질 전환된 사철느타리버섯으로부터 초원심분리 및 설탕밀도 기울기법으로 44% 층에서 미토콘드리아를 분리정제하였다. ATPase와 ATP synthase의 최적 활성조건은 각각 pH 7.4, $60^{\circ}C$ 및 pH 7.5, $57^{\circ}C$였고, km값은 11.6mM과 8.4mM였다. ATPase는 기질농도 5~6mM의 ATP에서, ATP synthase는 5~10mM ADP 농도에서 활성이 높으며, 그 이상의 농도에서는 기질 저해를 받았다. ATPase/ATP synthase 모두 $Mg^{2+}$ 의존성 효소로 $G_{418}$으로 비경쟁적인 저해를 받았다. 효소의 아미노산 분석결과 hydrophobic 아미노산 잔기는 50.5%, small 아미노산 잔기는 56.1%, hydrogen bonding 아미노산 잔기는 43.7%, helix breaking 아미노산 잔기는 55.2%였다. 인지질을 분석한 결과 phosphatidyl glycerol, phosphatidyl choline 및 phosphatidyl ethanolamine으로 구성되었고 phosphatidyl serine과 phosphatidyl inositol은 전혀 없었다. 포화 지방산은 palmitate(51.31%)와 stearate(18.32%)의 함량이 많았고, 불포화 지방산($C_{18:1}$, $C_{18:2}$ 및 $C_{16:1}$)의 함량도 많았다. The mitochondrion was purified at 44% sucrose layer from pleurotus florida by using ultracentrifuge and sucrose density gradient method. Optimum pH and temperature of ATPase and ATP synthase were pH 7.4, $60^{\circ}C$ and pH 7.5, $57^{\circ}C$ respectively, also their Km values were determined as 11.6mM and 8.4mM. ATPase was activated at 5~6mM ATP substrate concentration, then ATP synthase was 5~10mM range ADP. ATPase/ATP synthase were $Mg^{2+}$-dependent enzyme, partially inhibited by their substrate, and then showed an none competitive inhibition pattern by $G_{418}$. Amino acid composition of ATPase/ATP synthase was as follows, hydrophobic amino acid residue was 50.5%, small residue, 56.1%, hydrogen bonding residue, 43.7% and helix breaking residue, 55.2%. Phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl glycerol were contained but not phosphatidyl inositol and phosphatidyl serine. Palmitate(51.31%), stearate(18.32%) and unsaturated fatty acids($C_{18:1}$, $C_{18:2}$ and $C_{16:1}$) were predominated.

Purification and Characterization of Carboxypeptidase B from Wild Salmon (Salmo Salar) Eggs

김재웅,민태진,Jae Woong Kim,Tae Jin Min Korean Chemical Society 1985 대한화학회지 Vol.29 No.3

연어(Salmo Salar)알 중의 Carboxypeptidase B를 CM-셀룰로오스, 0.5포화황산 암모늄, DEAE-셀룰로오스 및 세파덱스 G-75젤로 정제하여 그 성질을 조사하였다. 이 효소의 최적 온도는 55$^{\circ}C$였으며, 최적 pH는 4.0과 7.0이었고, pH안정성은 2.0∼3.0 및 5.5∼7.0이었다. 히푸릴-L-아르기닌 기질에 대하여 글리실-L-아르기닌 부위를 절단하는 특이성을 보였고, 그 K$_m$값은 0.21mM이었다. Cu$^{2+}$ 와 Fe$^{3+}$ 존재하에서는 효소의 활성도가 증가하였지만 Zn$^{2+}$의 경우에는 감소하였다. 특히 리신은 히푸릴-L-아르기닌 기질에 대하여 경쟁적 억제작용을 보였으며, K$_i$값은 4.3mM이었다. 분자량은 36,400돌톤이었고, 19종류의 아미노산으로 구성된 단위체이었다. Carboxypeptidase B from Salmo Salar eggs was purified by CM-cellulose, 0.5 ammonium sulfate saturation, DEAE-cellulose, and Sephadex G-75 gel filtration and its enzymatic properties were investigated. Optimum temperature was 55$^{\circ}C$, pH optima were 4.0 and 7.0 at 37$^{\circ}C$, and the enzyme was stable at pH 2.0∼3.0 and 5.5∼7.0 for 1.5h. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-arginine. Its K$_m$ values was 0.21mM, and the enzyme activity was stimulated by Cu$^{2+}$ and Fe$^{3+}$, while inhibited by Zn$^{2+}$. The lysine was found to be competitive inhibitor and its K$_i$ value was determined to be 4.3mM. Molecular weight of this enzyme was determined to be 36,400 daltons by SDS-PAGE and the enzyme was monomeric protein composed of 19 kinds of amino acid residues.

복령 ( Poria cocos ( Schw . ) Wolf ) 중의 Carboxyl Proteinase 의 분리정제 및 그 성질에 관한 연구 ( 1 )

민태진,정광식,김재웅 ( Tae Jin Min,Kwang Sik Chung,Jae Woong Kim ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.3

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℃, pH stability, 1.2∼2.5 and thermal stability, 20°∼75 ℃. 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 0.29 mM when carbobenzoxy-L-tyrosine were used as a subtrate.

흰느타리버섯 ( Pleurotus cornucopiae ( Per . ) Rolland ) 중의 Protease 의 분리정제 및 성질에 관한 연구 ( 2 )

민태진,이수용,김재웅 ( Tae Jin Min,Soo Young Lee,Jae Woong Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2

The enzymic properties of protease in the Pleurotus cornucopiae(Per.) Rolland were investigated. The Km 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 SO₃^(-2), SO₄^(-2) and HP₄^(-2) but increased with NO₂^(-2) 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.

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.

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.

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.

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

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.