Histone 에 관한 연구 ( Ⅶ ) 밤나무 ( Castania crenata S . et Z . ) 꽃가루의 Histone

이희성,황운용,이경효,이근배 ( Hi Sung Lee,Woon Yong Hwang,Kyoung Hyo Lee,Keun Bai Lee ) 생화학분자생물학회 1976 BMB Reports Vol.9 No.2

It is now generally accepted that there are five main histones in the somatic cells of most species examined. However, some changes in the molecular species of animals and plants during the evolution have been reported. Recently, DeLange et al. (1971) discovered that there is a striking conservation and divergence of sequence during evolution for some of the histones between calf and pea bud. In this respect, it will be worth studying the histones of plant pollens which are unique in some species or class. We have studied on histones of some plant pollens from chestnut, pumpkin and pine tree which are distinctly related families each other. In the present studies, we report the results obtained from chestnut pollens. 1. The yield of whole histone recovered was 8.59 ㎎ per 1 g of pollens. This is very large amount as compared to those of mammary tissues. 2. The yield of DNA was 8.13㎎ per 1 g of pollens. Consequently the DNA to histone ratio was 1:1.06. 3. The relative amounts of five fractions, i.e., H1, H2a, H2b, H3 and H4 were 17.23, 19.32%, 26.89%, 23.06 and 13.50, respectively. 4. The electrophoretic mobility of individual histone fractions gave almost similar patterns to those of corresponding fractions of calf thymus. 5. We found that histone H2b fraction of chestnut pollens contained detectable amounts of ε-N-monomethyllysine. No evidence for the presence of methylated lysine or other side-chain derivatives was reported on this histone fraction. 6. Comparison of amino acid compositions reveals marked quantitative differences between corresponding chestnut pollens and pea bud histone fractions.

Histone 에 관한 연구 ( Ⅷ ) 호박 ( Cucurbita moschata Duch . ) 꽃가루의 Histone

이희성,장성길,라석찬,이근배 ( Hi Sung Lee,Sung Kil Chang,Suck Chan Rha,Keun Bai Lee ) 생화학분자생물학회 1976 BMB Reports Vol.9 No.2

Although studies on the histones from various kinds of animal sources have been reported, information about pollens is not available yet. The present report describes the properties of histones of pumpkin pollens. The results obtained were as follows: 1. The yield of whole histone was 12.2 ㎎ per g of pollens. 2. The yield of DNA was 9.1㎎ per g of pollens. Consequently, the DNA to histone ratio was 1 : 1.34. 3. The relative amounts of five histone fractions, i.e., Hl, H2a, H26, H3 and H4 were 12. 46%, 15. 16%, 27, 38%, 21. 23% and 23, 77%, respectively. The data presented in this report show that there is considerable variation in the relative amounts of histone fractions as compared to those of calf thymus. 4. Amino acid composition of individual histone fractions showed that there are marked quantitative differences between pumpkin pollens and pea bud. 5. Usually the methylated derivatives of lysine have been reported in various histone fractions of animal and plant sources. However, we found none o the methylated lysine residues in all the histone fractions from pumpkin pollens. 6. The electrophoretic mobility of individual histone fractions gave similar patterns to those of corresponding fractions of chestnut pollens.

Histone 에 관한 연구 ( 3 ) 정상인의 요속의 histone

이희성,이근배 ( Hi Sung Lee,Keun Bai Lee ) 생화학분자생물학회 1973 BMB Reports Vol.6 No.3

The histones have been precipitated from concentrate of pooled urine of healthy male middle school students by adding ammonium sulfate to 60% saturation and further fractionated by ethanol and acetone precipitation. A total of 71.5 ㎎ of total histone was recovered from 24ℓ of urine. The resulting four major fractions have all been characterized by analysis of amino acid composition and by polyacrylamide gel disc electrophoresis. The results established that the striking consistency did exist through all the corresponding fractions between urine sample and thymus and liver of calf. This analytical evidence suggests that the histones from human urine and calf thymus and liver seem to be homogeneous protein. The relative amounts of four major fractions, i.e., f1, f2 a, f2 b, and f3 were 21.8%, 24.5%. 22.5% and 31.2%, respectively. The amounts of fractions f1 and f3 from urine were almost similar to those of corresponding fractions of calf thymus and liver, marked increase in the amount of f2 a and concomitant derease in the amount of f2 b from urine were observed. The mechanism of occurrence of histones in urine and the physiological significance of histone excretion in urine remain to be clarified.

인삼의 유효성분에 관한 생화학적 연구(XII) 글루탐산-옥살초산 트란스아미나아제 및 글루탐산-피루브산 트란스아미나아제 활성에 미치는 인삼 성분의 영향(1)

김태봉,이희성,이근배,Kim, Tae-Bong,Lee, Hee-Sung,Lee, Keun-Bai 생화학분자생물학회 1977 한국생화학회지 Vol.10 No.4

인삼의 에탄올 추출물(WGpet-alc)은 사람 혈청 속에 있는 글루탐산-옥살초산 트란스아마나아제(GOT) 및 글루탐산-피루브산 트란스아미나아제(GPT)의 활성을 증가시키는 효과를 가졌으며, 이러한 효과는 WGpet-alc의 농도가 0.50% 일때 가장 현저하였고, 농도가 이보다 클 때는 도리어 감소되었다. 이와는 반대로, GOT 및 GPT의 활성은 인삼의 석유에테르 추출물 (WGpet)에 의해 크게 감소된다는 것이 확인되었다. Ethanol extract of Ginseng(WGpet-alc) was observed to increase the activities of human serum GOT and GPT in vitro and it was found that both enzymes showed the maximum activity when the concentration of WGpet-alc was 0.5% and the enzyme activity was decreased as the concentration of WGpet-alc rised over 0.5%. It was also observed that petroleum ether extract of Ginseng (WGpet) showed even at very low concentration an inhibitory effect on both enzymes.

종양조직의 Transfer Ribonucleic Acid의 Methyl화에 관한 연구

이근배,이희성,이강석,김기수,Lee, Keun-Bai,Lee, Hi-Sung,Lee, Kang-Suk,Kim, Ki-Sao 생화학분자생물학회 1971 한국생화학회지 Vol.4 No.1

정상조직으로는 동물의 간, 종양조직으로는 Sarcoma 180 ascites tumor cells 및 Walker 256 carcinosarcoma 등을 시료로 사용하여 tRNA를 정제 하였고 또 같은 조직에서 methylase를 추출하여 in vitro에서 tRNA를 homologous 및 heterologous하게 methyl화를 행하여 다음과 같은 몇가지의 결과를 얻었다. 1. Homologous하게 methylate 시켰을 때 종양조직이 정상조직 보다 3 배의 methyl화 능을 나타냈다. 2. 종양조직의 methylase로 정상조직의 tRNA를 기질로 하여 heterologous 하게 methyl화 시켰을 때 정상조직의 homologous methylase에 비하여 4-5배의 methyl 화능을 나타냈다. 특히 Walker 256 carcinosarcoma의 methylase의 활성도가 가장 높았다. 3. 종양조직에 존재하는 tRNA methylase는 guanine 유도체 보다 adenine 유도체를 더 많이 methylate 시켰다. 4. Paper chromatography로 분리 한 methylated bases는 14종이며 그 중 13종의 화학구조가 확인 되었다. 5. 정상조직 및 종양조직에서 문헌에 보고되어 있지 않은 N,N'-dimethylcytosine을 종양조직인 Walker 256 carcinosarcoma에서 분리 확인 하였다. 이 물질은 Walker 256 carcinosarcoma의 methylase로서 다른 종의 tRNA를 methyl화 시켰을 때에도 분리 확인 할 수 있었다. 6. Adenine 유도체로 추정되는 1종류의 methylated bases를 종양조직인 Walker 256 carcinosarcoma 및 Sarcoma 180 ascites tumor cells에서 각각 분리 하였다. Studies on transfer ribonucleic acid (tRNA) methylases of normal tissues, Walker 256 carcinosarcoma and Sarcoma 180 ascites tumor cells have been carried out with respect to: 1) rate of methylation of tRNA from rabbit liver, Walker 256 carcinosarcoma, Sarcoma 180 ascites tumor cells and E. coli Band 2) an attempt was also made to search out the pattern of methylation, i.e., the specific bases methylated by the extracts of tumor tissues. The homogenate was centrifuged at 100,000 xg for 10 min and the supernatant fluid was used as the enzyme extracts. In vitro methylation of tRNA was carried out by incubating enzyme extract with substrate (tRNA) and ATP generating system. $^{14}C$-methyl-L-methionine was used as methyl donor. Resulting methylated tRNA was hydrolyzed and chromatographed on Whatman paper. Spots on the chromatograms were identified by measuring the Rf and UV absorption spectra and cut into strips and counted directly in a Beckman LS-100 liquid scintillation counter. The results obtained were as follows: 1. The tRNA methylases of tumor tissues exhibited the elevaton of methylating potencies about three-fold increase when methylated homologously. 2. The methylase activity of tumor tissues was increased 4-5 times over that of normal controls. The enzyme extracted from Walker 256 carcinosarcoma served as the most appropriate source. 3. Several lines of evidence for the binary nature of the tRNA methylase from tumor tissues were demonstrated. Results of heterologous methylations with a variety of substrate reveal that extracts of tumor tissues introduce more methyl group into adenine residue than into guanine residue of tRNA. 4. Exposure of tRNA to normal methylases followed by methylases from tumor tissues has indicated that the tumor tRNA methylases introduced methyl groups into different positions on the tRNA molecule than do normal enzymes. Among hydrolysate of methylated tRNA, a novel methylated pyrimidine, i.e., N,N'-dimethylcytosine and an unidentified base were detectable. The only extract from Walker 256 carcinosarcoma was found to possess methylase activity for the synthesis of N,N'-dimethylcytosine.

인삼의 유효성분에 관한 생화학적 연구(XI) L-글루탐산-데히드로게나아제 활성에 미치는 인삼 성분의 영향 (2)

김태봉,이희성,이근배,Kim, Tae-Bong,Lee, Hee-Sung,Lee, Keun Bai 생화학분자생물학회 1977 한국생화학회지 Vol.10 No.3

사람 혈청 속에 있는 L-글루탐산-데히드로게나아제 (GlDH) 및 순수한 GlDH (Sigma사 제품)의 활성은 인삼의 에탄올 추출물(WGpet-alc)에 의해 다같이 증가되었지만, 인삼의 석유에테르 추출물(WGpet)에 의해서는 작은 농도에서도 감소되었다. 이러한 효소활성 증가효과 또는 감소효과는 흰쥐의 간 GlDH에 대한 실험결과와도 여러 점에서 공동된 양상을 나타낸다는 것이 확인되었다. On the basis of our previous report on the biochemical studies of Ginseng substances, it was attempted to observe the effect of ethanol extract (WGpet-alc) and petroleum ether extract (WGpet) of White Ginseng on the activity of human serum GlDH and pure GlDH in vitro. The activity of human serum GlDH and pure GlDH was significantly increased in the presence of WGpet-alc. On the other hand, an inhibitory effect of WGpet on GlDH was observed even at low concentration. It was also confirmed that these activating and inhibitory effects have the same pattern as the effects on albino rat hepatic GlDH.

사람 태반의 Branched Chain Amino Acid Transaminase에 관한 연구

박연수,이희성,이근배,Park, Yon-Soo,Lee, Hi-Sung,Lee, Keun-Bai 생화학분자생물학회 1978 한국생화학회지 Vol.11 No.4

사람 태반의 branched chain amino산 amino기 전위 효소에 관하여 관찰하였다. 이 효소는 세포질과 mitochondria에서 발견되며 세포질에 약 70~80%, mitochondnia에 8~15%가 분포되어 있으며 나머지 약간량이 핵분획에 들어 있다. 이 효소의 태반속의 활성도는 다른 조직 즉 흰쥐의 장, 취장 및 심장 등에 비하여 낮다. 이 효소는 세포질 속에서 2종의 isozyme 즉 Enzyme I과 Enzyme III이 발견되며 Enzyme I은 DEAE-cellulose column chromatography에서 인산염 완충액 0.02M에서 Enzyme III은 0.2M에서 각각 용출되며 Enzyme I은 활성도가 Enzyme III의 활성도보다 훨씬 높다. A study was undertaken on the distribution of branched chain amino acid aminotransferase (EC 2.6.1.6) in human placenta. This enzyme is localized in both the cytosolic and mitochondrial fraction. About 70~80 per cent of the total activity was found in the cytosolic fraction, 8~15 per cent in the mitochondrial fraction. In the human placenta the aminotransferase activities for branched chain amino acids are relatively low compared with those in rat tissues, i. e., intestine, pancreas, and heart. The present studies demonstrated that there are tow types of isozyme in the cytosolic fraction, Enzyme I and Enzyme III. Enzyme I was eluted by 0.02M phophate buffer from DEAE cellulose column and catalyzed the transamination of all three branched chain amino acids. Enzyme IQ was eluted by 0.2M buffer. It was found that the cytosolic fraction of human placenta contained much more Enzyme I than Enzyme III.

Purification and Properties of Two Glyoxylate Reductases from Germinating Soybean Seed Embryos

이근배,이희성,이강석,Lee, Keun-Bai,Lee, Hi-Sung,Lee, Kang-Suk 생화학분자생물학회 1970 한국생화학회지 Vol.3 No.2

발아중인 대두의 배에서 두 종류의 glyoxylate reductase(glycoIIate-NAD oxidoreductase, EC. 1. 1. 1. 26)를 추출하고 ammonium sulfate에 의한 침전, Sephadex G-200 gel filtration, DEAE-cellulose 및 Sephadex G-100 column chromatography에 의하여 정제하였다. 두 종류의 효소는 각각 NADH 및 NADPH를 cofactor로 요구하는 바 각각 745배 및 789배 정제하였다. 최종적으로 분리 정제된 상기 효소는 sucrose density gradient centrifugation, starch 및 acrylamide gel electrophoresis를 행하여 단일 효소 단백임을 확인하였다. NADH-linked glyoxylate reductase의 Michaelis constant, Km은 $9.0{\times}10^{-3}M$ 이다. 또한 NADH-Iinked enzyme 및 NADPH-linked enzyme의 분자량은 Sephadex G-100 gel filtration으로 측정하여 각각 142,000 및 192,000 이었다. 이 효소의 최적 pH, 최적온도, thiol group 저해제 및 몇 가지의 금속 ion이 효소활성에 미치는 영향을 검토하였다. Two protein fractions which catalyse the reduction of glyoxylate to glycollate were isolated from germinating soybean seed embryos by ammonium sulfate precipitation, Sephadex G-200 gel filtration. DEAE-cellulose column chromatography and rechromatography on Sephadex G-100. One protein fraction required NADH and another protein fraction required NADPH as cofactor. These two enzymes were purified 745-fold and 789-fold, respectively. The final preparations were homogeneous by sucrose density gradient centrifugation, starch and acrylamide gel electrophoresis. The apparent Michaelis constant. Km, of NADH-linked glyoxylate reductase was estimated as approximately $9.0{\times}10^{-3}M$. Molecular weights of NADH-Iinked and NADPH-linked enzymes were estimated to be about 142,000 and 192,000, respectively, by Sephadex G-100 gel filtration. Effects of pH, temperature, thiol group inhibitors and various metal ions on the enzyme were also described.

송아지 치수의 Histone

이근배,이희성,이강석,Lee, Keun-Bai,Lee, Hi-Sung,Lee, Kang-Suk 생화학분자생물학회 1973 한국생화학회지 Vol.6 No.2

저자들은 송아지 치수에서 histone을 추출 분리하였다. Histone의 추출 및 분리는 Johns 및 Johns and Phillips 등의 방법으로 분리 하였으며, 각 histone 분획들은 15% polyacrylamide disc gel 전기영동에 의하여 순도 및 mobility를 다른 조직들의 그것과 비교 검토하였다. 즉 흰쥐 및 마우스의 간, 송아지의 흉선 및 Sarcoma 180 ascites tumor cells의 histone과 비교 및 약간의 고찰을 시도하였으며, 다른 사람의 업적을 인용하여 비교하였다. 1. 송아지 치수에서 histone 주분획인 f1, f1a1, f2a2, f2b 및 f3 분획을 분리하였다. 2. Histone의 수율은 조직 1g 당 6.68mg 이다. 3. 각 분획의 polyacrylamide disc gel 전기영동으로 mobility는 다른 조직의 histone과 거의 같았다. 4. 송아지 치수의 histone 분포는 송아지 흉선의 그것에 비하여 많은 차이가 있다. 즉 f3 분획이 전체 histone의 39%로 가장 많았고, f2a1 및 f2a2 분획은 각각 6% 및 10.8%로 가장 적었으며, 각 분획의 함량은 다른 조직의 그것에 비하여 많은 차이가 있다. 5. 각 histone 분획의 amino 산 조성은 다른 조직의 그것과 별차이가 없다. Whole histone and histone fractions from calf dental pulp have been studied. Histones were extracted and fractionated according to the method of Johns and Phillips. The histone fractions were further purified by a polyacrylamide gel electrophoresis. The yield of whole histone recovered was 668 mg per 100 g of dental pulp. This is very large amount as compared to the calf thymus and liver. However, nearly similar amount was obtained from transplantable ascites carcinoma cells. Quantitative differences were found between a number of corresponding histone fractions of calf dental pulp and thymus. Striking low contents of fraction f2a1 (arginine-rich histone) were observed in dental pulp, being about one third of those of calf thymus. Fraction f2a2 was also significantly decreased. However, slightly higher contents of histones were found in fractions f1 and f3 plus f2b. The electrophoretic behaviors were compared with those of histones from calf thymus and rat liver. Amino acid analysis of the individual histone fractions showed that the over-all compositions were remarkably similar to those of corresponding fractions from calf thymus. The data of comparable histone fractions of calf and rat isolated by other workers have been included for comparison. The evidence for the tissue-specific differences in the distribution of the histone fractions among different tissues of the single animal was presented.

골수의 Polyol 및 Sugar Dehydrogenase에 관한 연구

안병문,이희성,이근배,Ahn, Byoung-Moon,Lee, Hi-Sung,Lee, Keun-Bai 생화학분자생물학회 1979 한국생화학회지 Vol.12 No.1

토끼 골수의 homogenate를 분별원심 분리하여 세포질, mitochondria 및 핵분획을 분리하여 sorbitol dehydrogenase, adonitol dehydrogenase 및 mannitol dehydrogenase의 분포를 관찰하였으며 aldose 인 여러 당류에 대한 sugar dehydrogenase의 존재유무를 관찰하여 다음과 같은 결과를 얻었다. 1. Sorbitol을 D-fructose로, D-fructose를 sorbitol로 전환시키는 sorbitol dehydrogenase는 골수에 있어서는 D-fructose를 sorbitol로 촉매하는 작용만을 하며 mitochondria에만 존재한다. 2. Adonitol(ribitol) dehydrogenase의 활성도는 조직 1g 당 약 196 unit로 sorbitol dehydrogenase 및 mannitol dehydrogenase 보다 2.6배 및 41배 높았으며 세포질, mitochondria 및 핵분획에 각각 분포되어 있다. 3. Mannitol dehydrogenase는 조직 1g 당 4.78 unit로 활성도가 낮으며, 이 중 약 88%인 4.2 units가 세포질에 함유되어 있다. 4. Sugar dehydrogenae는 기질에 따라 특이성을 나타냈다. 즉, 조직 1g 당 효소활성도는 rhamnose>xylose>lactose>glucose>mannose>galactose>maltose의 순이며 rhamnose는 활성도가 20.14 unit/g으로 가장 높았고 trehalose에 대한 활성도는 전혀 없었다. Polyol dehydrogenases, i.e., sorbitol dehydrogenase, adonitol dehydrogenase and mannitol dehydrogenase of rabbit bone marrow were studied. Bone marrow was homogenized and the distribution of the enzyme activity in cytosolic, mitochonrial and nuclear fractions was measured. Sugar dehydrogenase activity in the crude preparations was also observed. The results obtained were as follows: 1. Sorbitol dehydrogenase activity which reduces D-fructose in the presence of NADH to D-sorbitol was localized in the mitochondria) fraction. The reverse reaction was inactive in both cytosolic and mitochondria) fractions. 2. Adonitol (ribitol) dehydrogenase activity (196 units/g wet tissue) was found to be approximately 3 times higher than that of sorbitol dehydrogenase and 40 times higher than mannitol dehydrogenase. 3. Mannitol dehydrogenase activity was very low (4.2 units/g wet tissue) and 88% of total activity was found in cytosolic fraction. 4. Sugar dehydrogenase were also examined in the crude preparation of bone marrow. he enzyme was most active with rhamnose (20.14 units/g wet tissue), but a number of other sugars was also good substrate. Dehydrogenation was effective in the following order of activity: rhamnose>xylose>lactose>glucose>mannose>galactose>maltose Trehalose did not serve as substrate.