Molecular Structure of Phytochrome as Probed by Intrinsic Tryptophan Phosphorescence of Degraded 59 Kilodalton Chromopeptide

한태룡,송필순,Hahn, Tae-Ryong,Song, Pill-Soon Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.1

Phytochrome의 chromophore와 apoprotein의 분자 구조 및 배열을 알아보기 위해 분해된 small phytochrome (59 kD)의 트립토판 인광을 측정하였다. 인광수명값은 비교적 짧았으며 Pr형 (0.21초)과 Pfr형 (0.19초)간의 차이는 거의 없었다. 또한 트립토판에서 chromophore로의 에너지 전달은 관찰되지 않았다. 이는 large phytochrome (118 kD)에서 트립토판으로부터 Pr chromophore로 에너지가 효과적으로 전달된다는 사실 (Sarkar & Song. 1982)과는 상반된다. 이상의 결과로부터 proximal 트립토판잔기가 55 kD nonchromophore domain에 존재하며 이 부분이 단백분해 효소에 의해 잘려나가 small phytochorme 산불이 생성되는 것으로 믿어진다. The intrinsic phosphorescence of the degraded small phytochrome which has a molecular mass of 59 kilodalton(kD) was measured to probe the chromophore and apoprotein topography of phytochrome. Phosphorescence life times of tryptophan(Trp) residues of the small phytochrome show relatively short values and virtually no differences between the red light absorbing, Pr(0.21 s) and far red light absorbing, Pfr(0.19 s) forms. No delayed fluorescence from the chromophore upon excitation of Trp residues was observed, indicating that there is no energy transfer from the Trp(s) to the hromophore. The large phytochrome (118 kD) showed efficient energy transfer from the proximal Trp(s) to the Pr chromophore and a delayed fluorescence from the Pr hromophore upon excitation of Trps at 290 nm (Sarkar and Song, 1982). Results suggest that the 55 kD nonchromophore domain where the proximal Trp(s) resides is removed in the small phytochrome by proteolytic degradation.

분해된 59 kD chromopeptide 의 트립토판 인광측정에의한 phytochrome 의 분자구조

한태룡,송필순 ( Tae Ryong Hahn,Pill Soon Song ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.1

The intrinsic phosphorescence of the degraded small phytochrome which has a molecular mass of 59 kilodalton(kD) was measured to probe the chromophore and apoprotein topography of phytochrome. Phosphorescence life times of tryptophan(Trp) residues of the small phytochrome show relatively short values and virtually no differences between the red light absorbing, Pr(q.21 s) and far red light absorbing, Pfr(0.19 s) forms. No delayed fluorescence from the chromophore upon excitation of Trp residues was observed, indicating that there is no energy transfer from the Trp(s) to the chromophore. The large phytochrome (118 kD) showed efficient energy transfer from the proximal Trp(s) to the Pr chromophore and a delayed fluorescence from the Pr chromophore upon excitation of Trps at 290 ㎚ (Sarkar and Song, 1982). Results suggest that the 55 kD nonchromophore domain where the proximal Trp(s) resides is removed in the small phytochrome by proteolytic degradation.

RecA 중계 strand 교환에 대한 알칼화된 DNA 의 기질효과

한태룡 ( Tae Ryong Hahn ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.2

RecA protein catalyzes the homologous pairing and strand exchange between single stranded circular and homologous linear duplex DNA substrates forming nicked circular heteroduplex products. Effects of alkylated DNA substrates on RecA mediated strand exchange reaction were studied. Methylation of single stranded circular DNA by dimethylsulfate blocks strand exchange completely. Dimethylsulate reacts with amino groups of DNA bases to produce N-methylated products, which cannot form stable hydrogen bond. Thus pairing and strand exchange cannot occur between modified ssDNA and homologous intact duplex. In contrast to methylation, ethylated ssDNA by ethylnitrosourea reacts with homologous linear duplex to form stable nicked circular DNA products. However, the products were reduced with increase of degree of ethylation. When strand exchange was proceeded with ethylnitrosourea treated duplex DNA, similar results were obtained, although large amount of slow moving reaction intermediates were detected. Since less than 1% of nucleotides in DNA substrates is ethylated in the given reaction condition and most of the reaction products are ethylphosphotriesters (Singer et al., 1978), ethylated ssDNA may inhibit the formation of initiation complex, whereas ethylated dsDNA probably decrease the rate of homologous pairing and/or strand exchange.

Phytochrome 의 분자구조와 생리활성

한태룡(Tae Ryong Hahn),채쾌(Quae Chae) 생화학분자생물학회(구 한국생화학분자생물학회) 1988 생화학총설집 Vol.2 No.-

Biochemical and Pharmacological Study on The Bioactive Principles of Panax Ginseng

이현재,한태룡,김수자,Lee, Hyun-Jae,Hahn, Tae-Ryong,Kim, Soo-Ja 생화학분자생물학회 1979 한국생화학회지 Vol.12 No.2

인삼유효성분에 의한 강장 효과를 생체내 에너지 대사 과정과 연관시켜 새로운 생화학적 및 약리학적 견지에서 연구 검토하였으며, 이의 중요 결과는 다음과 같음. 즉 인삼의 메타놀 추출물은 토끼 근육으로 부터 유리한 효소인 creatine phospkokinase(CPKase)의 활성도를 증가시킴을 알았으며, 이러한 활성도의 증가 또는 활성화는 인삼 추출물의 농도에 따라 변화하되 약 5mg/ml 농도 이상에서는 최대 활성화율인 약 2배 정도로 유지됨을 알았다. 아울러 생체 투여 실험에 있어 생쥐에 인삼 추출물을 4주간 투여 (10mg/day)한 경우 심장 근육 조직 내의 phosphocreatine의 함량이 현저히 증가 되었으며 이런 현상은 동물을 36시간 동안 굶겼을 경우에도 대조군보다 높은 값을 유지됨을 알았다. 이러한 인삼추출물에 의한 효과는 세포내 에너지 대사과정과 밀접한 관계를 맺고 있다고 추정할 수 있었으며 인삼 유효성분의 주요 약효를 에너지 대사와 관계된 강장 효과로서 설명코져 시도하였음. The biochemical efficacy of Panax ginseng was studied with the methanolic extract of ginseng by a new biochemical approach employing a rabbit muscle creatine phosphokinase (CPKase, EC 2.7, 3.1). The study included both effects of ginseng extract on the in vitro activity of CPKase and on the tissue levels of phosphocreatine and ATP by in vivo administration. In this study, the activity of CPKase was measured in both directions of its reversible reactions with the corresponding coupled enzyme systems, and it was demonstrated that ginseng extract causes a marked acceleration in the rate of the in vitro activity of CPKase up to about two fold and shows a hyperbolic saturation curve in its concentration dependent relative activity profile of CPKase. In addition, from the in vivo experiment with mice, it was also demonstrated that the administration of ginseng extract orally causes an increase in the tissue level of phosphocreatine and thus prevents the decrease in the tissue content of phosphocreatine which goes along with a period of fasting the animals. From the results, it was also suggested that ginseng extract may affects on the energy-linked cellular metabolic reactions by promoting the activity of CPKase and thus causes an efficient utilization of high-energy phosphates for many cellular metabolic activities under the stressful conditions.

분리된 완두 엽록체 Fructose - 1 , 6 - bisphosphatase 의 생화학적 특성

조만호,한태룡 ( Man Ho Cho,Tae Ryong Hahn ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

Chloroplast fructose- 1,6-bisphosphatase from pea leaves was purified and characterized. Monomeric molecular weight of the enzyme was estimated as 48,000 by SDS polyacrylamide gel electrophoresis. Molecular weight of the intact enzyme as determined by Sephadex G-200 gel permeation chromatogrphy was about 160,000. Thus the enzyme is believed to be tetrameric and composed of four identical subunits. The purified chloroplast enzyme was inactive at neutral pH but active at alkaline pH. The enzyme was also activated by Mg^(2+) and reducing agents such as dithiothreitol. Gluconeogenic fructose-1,6-bisphosphatase in cytosol is known to be active at neutral pH and insensitive to Mg^(2+) and reducing agents. The enzyme was insensitive to AMP which is a potent inhibitor for cytoplasmic fructose-1,6-bisphosphatase. The saturation curve of Mg^(2+) shows sigmoidal shape and Hill coefficient was 3.3. Activation energy (E_a) and Arrhenius frequency factor (A) of the enzyme catalyzed reaction were 21.5 kcal/mol and 4.01×10^8/s, respectively. Activation entropy (△S^(≠0) and activation enthalpy (△H≠0) of the enzyme reaction were -19.17 cal/K·mol and 20.87 kcal/mol, respectively.

형질전환된 E . Coli LE392 로 부터 Plasmid - encoded thioredoxin 의 분리정제 및 특성

조만호,한태룡 ( Man Ho Cho,Tae Ryong Hahn ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.1

치자 Genipin과 아미노산의 청색소변환반응에 관한 물리화학적 연구

이재연,한태룡,백영숙,Lee, Jae-Youn,Hahn, Tae-Ryong,Paik, Young-Sook 한국응용생명화학회 1998 Applied Biological Chemistry (Appl Biol Chem) Vol.43 No.4

우리 나라에서 오랫동안 식용 및 황색 색소원으로 이용되어 온 치자(Gardenia jasminoides)열매로부터 iridoid glycoside인 geniposide를 분리, 정제한 후 ${\beta}-glucosidase$로 가수분해하여 얻은 genipin을 glycine, alanine, histidine, lysine, phenylalanine, glutamate 등 여섯 종류의 아미노산과 반응시켜 수용성 치자청색소로 전환되는 과정을 규명하였다. Genipin이 아미노산과 반응하여 청색소가 되는 과정에서 pH의 영향을 알아보기 위하여 여러 pH에서 반응을 시켜본 결과 청색소 생성의 최적조건은 pH 7.0 이었고, pH 3.0 조건에서는 청색소가 전혀 생성되지 않았으며, pH 12.0 조건에서는 미량의 청색소만 생성되었다. 아미노산의 종류에 따라서도 청색소 생성량 및 색감에 차이가 있었는데 $Iysine({\lambda}_{max}=573\;nm),\;glycine({\lambda}_{max}=595 \;nm),\;phenylalanine({\lambda}_{max}=602\;nm),\;alanine({\lambda}_{max}=595\;nm)$에 비해 $histidine({\lambda}_{max}=601\;nm)$과 $glutamate({\lambda}_{max}=601\;nm)$의 경우에는 비교적 적은 양의 청색소가 생성되었다. 청색소 생성 속도상수를 여러 온도$(60,\;70,\;80,\;90^{\circ}C,\;pH\;7.0\;phosphate$ 완충용액)에서 구하였는데, 염기성 아미노산이 중성 및 산성 아미노산에 비해 생성속도가 빨랐다. 이들 값으로부터 Arrhenius 활성화에너지를 계산한 결과 $glycine(E_A=9.8\;kcal/mol)$이 다른 아미노산$(E_A=13.3{\sim}15.4\;kcal/mol)$에 비해 특히 작은 값의 활성화에너지를 나타내었다. Genipin was obtained from hydrolysis of geniposide isolated from gardenia fruits with ${\beta}-glucosidase$. Reaction of genipin with glycine, alanine, histidine, lysine, phenylalanine and glutamate in aqueous buffer solution converted colorless starting materials to blue pigments. Effect of pH for the formation of blue pigments was tested using UV/Vis spectrophotometer. The optimum pH for the formation of blue pigments was 7.0. No pigment and trace amounts were formed at acidic (pH 3.0) and alkaline (pH 12.0) conditions, respectively. The amount and tincture of blue color were distinct with different amino acids. In contrast with lysine $({\lambda}_{max}=573\;nm)$, glycine $({\lambda}_{max}=595\;nm)$, phenylalanine $({\lambda}_{max}=602\;nm)$ and alanine $({\lambda}_{max}=595\;nm)$, the reaction of genipin with histidine $({\lambda}_{max}=601\;nm)$ and glutamate $({\lambda}_{max}=601\;nm)$ produced relatively small amounts of blue pigments. Rate constants for the formation of blue pigments from genipin with amino acids at various temperatures $(60,\;70,\;80,\;90^{\circ}C,\;pH\;7.0\;phosphate\;buffer)$ were obtained. Rate constants of genipin with basic amino acids were larger than neutral or acidic amino acids. Arrhenius activation energies of the formation of blue pigments indicated that activation energy of glycine $(E_A=9.8\;kcal/mol)$ was especially lower than those of other amino acids $(E_A=13.3{\sim}15.4\;kcal/mol)$.

인삼 유효성분에 관한 생화학적 및 약리학적 연구

이현재,한태룡,김수자 ( Hyun Jae Lee,Tae Ryong Hahn,Soo Ja Kim ) 생화학분자생물학회 1979 BMB Reports Vol.12 No.2

The biochemical efficacy of Panax ginseng was studied with the methanolic extract of ginseng by a new biochemical approach employing a rabbit muscle creatine phosphokinase (CPKase, EC 2.7, 3.1). The study included both effects of ginseng extract on the in vitro activity of CPKase and on tle tissue levels of phosphocreatine and ATP by in vivo administration. In this study, the activity of CPKase was measured in both directions of its reversible reactions with the corresponding coupled enzyme systems, and it was demonstrated that ginseng extract causes a marked acceleration in the rate of the in vitro activity of CPKase up to about two fold and shows a hyperbolic saturation curve in its concentration dependent relative activity profile of CPKase. In addition, from the in vivo experiment with mice, it was also demonstrated that the administration of ginseng extract orally causes an increase in the tissue level of phosphocreatine and thus prevents the decrease in the tissue content of phosphocreatine which goes along with a period of fasting the animals. From the results, it was also suggested that ginseng extract may affects on the energy-linked cellular metabolic reactions by promoting the activity of CPKase and thus causes an efficient utilization of high-energy phosphates for many cellular metabolic activities under the stressful conditions.

완두엽록체 fructose - 1 , 6 - bisphosphatase 의 분리정제 및 특성

부성희,한태룡 ( Seong Hee Bhoo,Tae Ryong Hahn ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.4

Chloroplast fructose-1,6-bisphosphatase was purified 1,300 fold from pea leaves. The purified enzyme appeared homogeneous and the approximate molecular weight of the monomer was 40,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was inactive at pH 7.5, while it was activated by dithiothreitol or alkaline pH, indicating that the purified fructose-1,6-bisphosphatase was originated from chloroplast. The enzyme saturation curves with fructose-1,6-bisphosphate and Mg^(++) show sigmoidal shapes with almost same Hill coefficients (2.6 and 3.1, respectively), suggesting that the enzyme is composed of multimer. The substrate concentration required for half-maximal activity was 40 μM which is a comparable value (80μM) for spinach chloroplast fructose bisphosphatase (Zimmermann et al., 1976).