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 ( 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.

Studies on Histones from Sarcoma 180 Ascites Tumor Cells. Methylation and Acetylation

이희성,Lee, Hi-Sung 생화학분자생물학회 1971 한국생화학회지 Vol.4 No.2

Sarcoma 180 복수암 세포에서 histone의 여러분획 즉 f1, f2a1, f2a2, f2b 및 f3로 분리 정제하여 in vitro에서 acetylation 및 methylation에 관한 연구를 행하였다. Methyl화 및 acetyl화의 관찰은 Sarcoma 180 복수암 세포의 핵을 L-methionine-Methyl-$^{14}C$ 또는 sodium acetate-1-$^{14}C$ 존재하에 jn vitro에서 행하였으며 histone 분획을 f1, f2a1, f2a2, f2b, 및 f3 별로 각각 추출 정제하였다. Methyl화 된 histone 분획들을 산으로 가수분해한 후 amino acid analyzer로 각 ammo 산을 각각 분리 정제하여 methyl화 또는 acetyl화한 amino 산 유도체 들을 동정 및 정량하였다. Histone을 methyl화 시킨 결과 polypeptide chain의 histidine 및 lysine 잔기의 유리 $\epsilon$-amino group에 methyl근이 transfer 되었다. 각 histone 분획의 비활성은 f2a1이 가장 높았으며 f2b, f2a2, f3 및 f1의 순으로 방사능이 낮었다. $\epsilon$-N-MethylIysine의 방사능을 histone의 여러 분획 즉 f1, f2a1, f2a2 및 f2b에서 증명하였으며 $\epsilon$-N-dimethyllysine의 방사능이 $\epsilon$-N-monomethyllysine 보다 1.7배나 많었다. Histone 분획 f2a2와 f2b에서는 3-methylhistidine에서도 방사능을 계측할 수 있었다. Whole histone의 각 분획에서 계측된 방사능의 값은 그 분획에서 분리한 $\epsilon$-N-monomethyllysine, $\epsilon$-N-dimethyllysine 및 3-methylhistidine의 방사능의 합계와 일치하였다. Acetyl 화의 경우 각 histone의 분획들을 trypsine과 Pronase로 가수분해하여 얻은 peptide와 amino 산들을 exclusion chromatography 및 ion exchange chromatography로 분리하였다. Acetyl 화를 행하였을 때는 arginine-rich 분획인 f2a1, f2a2 및 f3에만 incorporate 되었다. Histone의 각 분획중 f2a1이 다른 분획에 비하여 방사능이 가장 높았으며 f2a2, f3, f2b 및 f1의 순으로 방사능이 낮어진다. 각 분획의 방사능은 그의 전부가 $\epsilon$-N-acetyllysine 및 ${\alpha}$-N-acetyllysine이 갖인 방사능에 기인하였다. Lysine-rich histone 분획인 f1과 f2b에는 acetylation이 일어나지 않았으며 따라서 $\epsilon$-N-acetyllysine 및 $\alpha$-N-acetyllysine을 검출할 수 없었다. In order to observe the properties of histones of Sarcoma 180 ascites tumor cells, the various histone fractions isolated from the tumor cells following the methylation and acetylation were studied. Histone fractions f1, f2a1, f2a2, f2b and f3 were prepared from isolated nuclei which had been incubated in the presence of L-methionine-methyl-$^{14}C$ and sodium acetate-1-$^{14}C$, respectively. For the methylation, the various histone fractions were hydrolyzed in acid and the hydrolysates were chromatographed on an amino acid analyzer. Histones were methylated by the transfer of methyl groups to the free $\epsilon$-amino groups of lysine and histidine residues in the polypeptide chain, after the histone molecules had been synthesized. The specific activity of the histone fraction f2a1 was greater than those of other fractions and the specific activities diminished in the order of f2b, f2a2, f3 and f1 fractions. Histone fractions f1, f2a1, f2a2 and f2b were found to contain radioactive $\epsilon$-N-methyllysine. $\epsilon$-N-Dimethyllysine was the predominant form of the modified amino acid and its concentration exceeds that of the $\epsilon$-N-monomethyllysine by a factor of 1.7. Histone fractions f2a2 and f2b were found to contain radioactive 3-methylhistidine. Virtually all of the radioactivity in these histone fractions are recoverable from the $\epsilon$-N-monomethyllysine, $\epsilon$-N-dimethyllysine and 3-methylhistidine. For the acetylation, each of the histone fractions was digested with trypsin and Pronase, and the resulting peptides and amino acids were separated by exclusion chromatography and ion exchange chromatography. Only the arginine-rich fractions f2a1, f2a2 and f3 were found to be appreciably labeled with acetate-$^{14}C$. Among the histone fractions, f2a1 fraction had a considerably higher specific activity than the other histone fractions and the specific activities diminished in the order of f2a2, f3, f2b and f1 fractions. All of their radioactivities could be recovered as two major radioactive peaks which were identified as $\epsilon$-N-acetyllysine and $\alpha$-N-acetyllysine. The comparatively lysine-rich histone fractions f1 and f2b were unlabeled and did contain $\epsilon$-N-acetyllysine and $\alpha$-N-acetyllysine.

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.

고속철도시스템의 기술이전을 통한 철도산업발전 방안

이희성(Hi Sung Lee),강태원(Tae Won Kang) 한국철도학회 2007 한국철도학회논문집 Vol.10 No.6

고속철도시스템 기술도입은 관련 철도산업을 발전시키고 타 산업에 파급효과를 주어 철도기술을 동반 발전시켰다. 이에 고속철도시스템의 기술 도입시의 기술이전 및 국산화 과정을 검토하고, 기술이전 및 국산화과정에서 나타난 문제점을 분석하였다. 그리고 철도시스템 기술을 유지 및 확대하기 위하여 기술이전을 통한 철도산업발전방안이 검토되었다. This paper suggests that the introduction of Seoul-Pusan High Speed rail system makes related railway industry to be developed through transfer of high technology and other related industries to be jointly grown by technology’ ripple effects. Technology transfer and localization implementation process in the period of technology introduction was reviewed synthetically and problems at that period was also analyzed. And technology development countermeasures of railway industry was discussed in order to maintain and expand railway system technology in the future.

Sarcoma 180 복수암세포 Histones 의 Methyl 화 및 Acetyl 화에 관한 연구

이희성 ( Hi Sung Lee ) 생화학분자생물학회 1971 BMB Reports Vol.4 No.2

In order to observe the properties of histones of Sarcoma 180 ascites tumor cells, the various histone fractions isolated from the tumor cells following the methylation and acetylation were studied. Histone fractions f1, f2a1, f2a2, f2b and f3 were prepared from isolated nuclei which had been incubated in the presence of L-methionine-methyl-^(14)C and sodium acetate-l-^(14)C, respectively. For the methylation, the various histone fractions were hydrolyzed in acid and the hydrolysates were chromatographed on an amino acid analyzer. Histones were methylated by the transfer of methyl groups to the free s-amino groups of lysine and histidine residues in the polypeptide chain, after the histone molecules had been synthesized. The specific activity of the histone fraction f2a1 was greater than those of other fractions and the specific activities diminished in the order of f2b, f2a2, f3 and f1 fractions. Histone fractions f1, f2a1, f2a2 and f2b were found to contain radioactive e-N-methyllysine. e-N-Dimethyllysine was the predominant form of the modified amino acid and its concentration exceeds that of the ε-N-monomethyllysine by a factor of 1.7. Histone fractions f2a2 and f26 were found to contain radioactive 3-methylhistidine. Virtually all of the radioactivity in these histone fractions are recoverable from the ε-N-monomethyllysine, ε-N-dimethyllysine and 3-methylhistidine. For the acetylation, each of the histone fractions was digested with trypsin and Pronase, and the resulting peptides and amino acids were separated by exclusion chromatography and ion exchange chromatography. Only the arginine-rich fractions f2a1, f2a2 and f3 were found to be appreciably labeled with acetate-^(14)C. Among the histone fractions, f2a1 fraction had a considerably higher specific activity than the other histone fractions and the specific activities diminished in the order of f2a2, f3, f2b and f1 fractions. All of their radioactivities could be recovered as two major radioactive peaks which were identified as ε-N-acetyllysine and α-N-acetyllysine. The comparatively lysine-rich histone fractions f1 and f2b were unlabeled and did contain ε-N-acetyllysine and α-N-acetyllysine.

곡선부 주행 차량의 마모특성에 관한 연구

이희성(Hi Sung Lee) 한국철도학회 2007 한국철도학회논문집 Vol.10 No.6

곡선통과 새마을호 열차의 마모 특성을 평가하기 위하여 다량 편성 해석모델이 사용된다. 이 모델과 ADAMS/Rail를 사용하여 마모 특성과 관련된 변수의 변화에 따른 민감도 해석이 수행된다. 저속에서 우측 차륜의 마모도와 미끄럼도가 좌측 차륜보다 높으나, 고속의 경우는 좌측 차륜이 우측 차륜보다 높다. 곡선반경의 감소에 따라 마모도와 미끄럼도가 증가한다. 완화곡선의 길이가 증가하면 마모도와 미끄럼도가 증가한다. 또한 캔트가 증가하면 마모도와 미끄럼도가 증가한다. For the wear characteristics assessment of Saemaul train passing through curves, an analysis model for multi-car system has been developed. By using this model and ADAMS/Rail, sensitivity analyses have been conducted for the wear characteristics by changing the related parameters. At low speed, the wear number and the sliding mean of right wheel showed higher than left wheel, while those of left wheel showed higher than right wheel at high speed. According to the decrease of curve radius, the wear number and the sliding mean were increased. When the length of transition curve was increased, the wear number and the sliding mean was increased. And according to increase of cant, the wear number and the sliding mean were increased.

Glyoxyl 산 환원효소의 Subunit ( 1 )

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

Glyoxylate reductase (glycollate-NAD oxidoreductase, 1. 1. 1. 26) has been prepared from 20,000 x g supernatant fraction of homogenate of germinating soy bean seed cotyledons and purified by ammonium sulfate precipitation, Sephadex G-200 gel filtration and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated to be about 158,000 by Sephadex G-100 gel filtration. The purified enzyme has been dissociated into a molecule one fourth the molecular weight of the native enzyme by treatment with 1 per cent sodium dodecyl sulfate, β-mercaptoethanol and Tris-buffer at pH 7.1 for 30 min. The molecular weight of the subunit was estimated to be approximately 41,500±4,000 by polyacrylamide gel electrophoresis. Amino acid composition of the above enzyme molecule are also presented. Some molecular properties of the subunit are being investigated.

Histone 에 관한 연구 ( Ⅴ ) Bakers ' Yeast 의 Histone

라석찬,이희성,이근배 ( Suck Chan Rha,Hi Sung Lee,Keun Bai Lee ) 생화학분자생물학회 1975 BMB Reports Vol.8 No.1

Although studies on histone fractions isolated from various kinds of animal $quot;sources$quot; have been reported, yeast was not studied except qualitative analysis by Rozijn and Tonino, and Duffus. In an attempt to investigate histone fractions of yeast, nuclei was isolated from a yeast, Sdccharomyces cerevisiae, by the procedure of Rozijn and Tonino, and histone fractions f1(KAP), f2a(GRK+LAK), f2b(KAS) and f3(ARE) were prepared from isolated nuclei by the procedure of Johns. The resulting four fractions have all been put on 15% polyacrylamide disc gel electrophoresis and chromatographed for analysis of amino acid composition. DNA was assayed by the method of Burton. The results of the experiments were summarized as follows: 1. The yield of whole histone was 3.257 mg per 1 g of yeast. This amount was very similar to calf thymus and liver. 2. The yield of DNA was 2. 916 mg per 1 g of yeast. The DNA to histone ratio was 1 : 1.117. 3. The relative amounts of four fractions, i. e., f1(KAP), f2a(GRK+LAK), f2b (KAS), and f3(ARE) were 46.6%, 1.4%, 49.4% and 4.9%, respectively. 4. The amounts of fractions f1, f2a, f2b and f3 from yeast were remarkably different to those of corresponding fractions of other source, marked increase in the amount of f1 and f2b, and marked decrease in the amount of f2a and f3 from yeast were observed. 5. The electrophoretic pattern of histone fraction gave similar pattern to those of calf thymus and mouse liver, and fractions f1, f2b and f3 were electrophoretically homogeneous in 15% polyacrylamide disc gels at pH 4.0. 6. Amino acid analysis of the individual histone fractions showed that the over-all compositions were similar but not identical to those of corresponding fractions from calf thymus and other mammalian cells.

Sn-Glycerol-3-Phosphate Dehydrogenase에 관한 연구 [V] Affinity Chromatography에 의한 사람 태반의 $\alpha$-glycerophosphate 탈수소 효소의 정제 및 성상에 관한 연구

황운용,이희성,이근배,Hwang, Woon-Young,Lee, Hi-Sung,Lee, Keun-Bai 생화학분자생물학회 1979 한국생화학회지 Vol.12 No.1

사람 태반조직에서 세포질에 있는 NAD 의존성 $\alpha$-Glycerophosphate 탈수소 효소(sn-glycerol-3-phosphate: $NAD_+$ 2-oxidoreductase EC 1.1.1.8)의 분포를 관찰하였으며 이 효소를 정제하였다. 또 mitochondria에 있는 FAD 의존성 $\alpha$-Glycerophosphate 탄수소 효소(sn-glycerol-3-phosphate: cytochrome oxidoreductase, EC 1.1.99.5)의 분포를 관찰하였다. 세포질의 NAD 의존성 효소의 활성도는 White and Kaplan 법으로 mitochondria에 있는 FAD 의존성 효소의 활성도는 Dawson and Thorne의 방법으로 각각 측정하였다. 사람 태반조직 1g당 NAD 의존성 효소의 활성도는 homogenate에서 14 unit이며 세포질에서는 13.3 unit로 세포질분획에 95%가 함유되어 있다. FAD 의존성 효소의 활성도는 homogenate에 약 33 unit이 며 mitochondria 분획에는 약 29 unit로 mitochondria 분획에서 약 87%가 회수되었으며 세포질에는 전혀 효소의 활성이 없었다. NAD 의존성 및 FAD 의존성 $\alpha$-glycerophosphate 탈수소효소의 비활성도는 각각 0.41 및 2.83으로 FAD 의존성 효소가 NAD 의존성 효소보다 비활성도가 약 7배 높았다. 세포질에 있는 NAD 의존성 이 효소를 ammonium sulfate에 의한 침전, blue dextran-Sepharose column chromatography 및 DEAE-cellulose column chromatography 등으로 655배 정제하였으며 polyacrylamide gel 전기영동으로 측정한 분자량은 61,500이다. 정제된 이 효소는 DEAE-cellulose column chromatogram상에 단일 peak의 효소활성을 나타낸 점으로 보아 single major isozyme으로 되어 있는 것 같다. 정제된 이 효소의 반응 최적 온도는 $55^{\circ}C$이며 반응 최적 pH는 9.5이다. NAD 의존성 이 효소의 $\alpha$-glycerophosphate 및 $NAD_+$에 대한 Km 값은 각각 3.6mM 및 0.38mM이다. 이 효소를 $55^{\circ}C$에서 10분간 처리하였을 때 효소 활성이 약 50%가 감소하였다. 이 효소는 비교적 열에 대하여 안정한 것 같다. $K^+$은 낮은 농도인 $1.0{\times}10^{-6}M$에서 NAD 의존성 효소의 활성을 약 75% 억제하였다. 그러나 $Na^+$은 $1.0{\times}10^{-6}M$에서 효소의 활성이 약 25% 억제되었으며 농도의 증가에 따라 점차적으로 더 억제되었다. $Ca^{++}$의 경우 $1.0{\times}10^{-6}M$ 농도에서 효소활성이 약 72% 억제되었으나 농도의 증가에 따라 억제현상이 감소됨을 알 수 있었다. $Mg^{++}$ 경우 $1.0{\times}10^{-6}M$에서는 효율활성이 약 43% 촉진되었으며 농도의 증가에 따라 효율활성이 억제되었다. p-Chloromercuribenzoate는 $1.0{\times}10^{-6}M$ 농도에서 정제된 NAD 의존성 효소의 활성을 약 25% 억제하였으며 농도의 증가에 따라 억제현상도 증가되어 $1.0{\times}10^{-2}M$에서는 약 88%가 억제되었다. The distribution and some properties of $\alpha$-glycerophosphate dehydrogenase of human placenta have been studied. Cytosolic NAD-linked $\alpha$-glycerophosphat dehydrogenase (sn-glycerol-3-phosphate : $NAD^+$ 2-oxidoreductase, EC 1.1.1.8) activity was assayed by the method of White and Kaplan. The activity in homogenate was found to be 19. units per g wet tissue and was recovered in cytosolic fraction(specific activity: ca. 0.4). The activity of mitochondrial FAD-linked $\alpha$-Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate : cytochrome oxidoreductase, EC 1.1.99.5) was measured by the procedure of Gonzalez-Cerozo and Dalziel. The activity in homogenate was 33 units and was recovered about 87 per cent in mitochondrial fraction(specific activity: 2.8). Thus, the specific activity of FAD-linked enzyme in mitochondria was found to be about seven-fold greater than that of NAD-linked enzyme in cytosol. Cytosolic NAD-linked $\alpha$-glycerophosphate dehydrogenase from human placenta has been purified about 655 fold by ammonium sulfate precipitation and affinity chromatography on blue dextran-Sepharose column and followed by DEAE-cellulose column chromatography. The elution profile of NAD-linked enzyme from cytosolic fraction was found to be a single major isozyme on the DEAE-cellulose column chromatogram. The molecular weight was estimated to be 61,500 by acrylamide gel elecarophoresis in the presence of dodecyl sulfate. The optimal pH was 9.5. Optimal temperature was 55, at this point, incubation for 10 min produced about 50 per cent inhibition of the enzyme activity. Data with prolonged heating suggested that the enzyme is relatively stable to thermal inactivation. p-Chloromercuribenzoate at a concentration of $10\;{\mu}M$ produced a 25 per cent inhibition of the enzyme and the increase in the concentration exhibited a gradual inhibition. $Na^+$ of $1.0{\mu}M$ concentration also exhibited about 25 per cent inhibition. The same concentration of $K^+$ showed more strong inhibition and the enzyme retained only 25 per cent of original activity. $Ca^{++}$ inhibited the enzyme to a slightly lesser extent. In contrast, the same concentration of $Mg^{++}$ stimulated about 40 per cent of the enzyme activity. However, the greater concentration showed inhibitory effect.