인체 α 1 - antitrypsin cDNA 의 분리 및 E . Coli 내에서의 발현

이상철,유명희 ( Sang Chul Lee,Myeong Hee Yu ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

Human α1-AT cDNA was screened from human liver cDNA library, by use of oligonucleotide probes. DNA insert of 1.3 kbp from a positive clone was subcloned into pUC9 plasmid and was analyzed by restriction enzyme digestion and partial DNA sequencing. The obtained gene was identical to human αl-AT cDNA reported previously. In order to express αl-AT cDNA under regulation of tac promoter, expression vector pT-AT was constructed. E. coli JM109 cells transformed with pT-AT showed αl-AT activity upon induction of recombinant protein synthesis. The recombinant αl-AT was immunologically identical to authentic αl-AT that was purified from human blood. When the recombinant cell lysates were analyzed on SDS-polyacrylamide gel electrophoresis, the expressed α1-AT was not detected by staining with Coomassie Brilliant Blue. However Western blotting of the same gel, using rabbit IgG against αl-AT and peroxidase-conjugated goat anti-rabbit IgG antibody, showed the existence of αl-AT at the expected molecular weight region. Molecular weight of recombinant αl-AT was slightly lower than that of authentic αl-AT; which presumably was due to lack of glycosylation inside E. coli cells.

Identification of Essential Cysteine and Arginine Residues in Glucose-6-phosphate Dehydrogenase from the Uropygial Gland of Duck

이상철,김유삼,Lee, Sang-Chul,Kim, Yu-Sam 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.2

오리의 uropygial gland로 부터 pI 6.6인 glucose-6-phosphate dehydorgenase를 순수하게 정제하여 효소활성과 기질 및 보조효소와의 결합에 관여하는 아미노산에 대해 조사하였다. Cysteine의 -SH기에 특이하게 반응하는 p-chloromercuriphenylsulfonic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide와 arginine의 guanidino기에 특이하게 반응하는 phenylglyoxal 등이 효소의 활성을 억제한다는 사실로 보아 효소의 active site나 그 근처에 cysteine과 arginine이 존재하는 것으로 예상된다. 또한 glucose-6-phosphate가 존재할 때 이 reagent들에 의한 효소의 활성억제에 아무런 영향이 없었으나 $NADP^+$존재하에서는 효소의 활성억제 현상이 크게 감소한다는 사실로 미루어 cysteme과 arginine이 $NADP^+$와 효소가 결합하는 위치냐 또는 그 부근에 있는 것으로 판단된다. $Mg^{++}$이 존재할 때 p-chloromercuriphenylsulfonic acid, 5,5'-dithio-bis(2-nitrobenzoic acid)와 phenylglyoxal에 의한 효소의 활성 억제가 더 크게 나타났는데, 이러한 사실은 EDTA와 $Mg^{++}$이 공존할 때 이 reagent에 의한 활성 억제가 EDTA가 존재하지 않을 때 보다 감소하였다는 것으로 보아 $Mg^{++}$이 이 reagent에 의한 효소의 활성 억제를 증가시킨다는 사실을 확인하였다. Glucose-6-phosphate dehydrogenase from the uropygial gland of duck was purified in an electrophoretically homogenous form and its essential amino acid for catalysis was investigated. Three -SH group directed reagents, p-chloromercuriphenylsulfonic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid) and N-ethylmaleimide, inhibited the enzyme activituy, and arginine directed reagent, phenylglyoxal, also inhibited the enzyme activity. The inhibition of the enzyme by these reagents was protected by addition of $NADP^+$ whereas it was not affected by that of glucose-6-phosephate indicating that essential -SH and guanidino group are located on the $NADP^+$ binding region of active site. $Mg^{++}$ promoted the inhibition of the enzyme by the -SH and guanidino group directed reagents. But the promotion of the inhibition was considerably recovered in the presence of EDTA. This results suggested that divalent metal cation may affect the conformational change toward the susceptible one against modifying reagents.

인체 ${\alpha}1$-antitrypsin cDNA의 분리 및 E.coli 내에서의 발현

이상철,유명희,Lee, Sang-Chul,Yu, Myeong-Hee 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

인체 간 cDNA library로부터 올리고 뉴클레오티드 프로브를 이용하여 ${\alpha}1$-antitrypsin (${\alpha}1$-AT) cDNA를 지닌 파지 클론을 스크린하였다. 선별된 클론에는 1.3 k bp의 DNA가 삽입되어 있었으며 이를 pUC9 벡터에 다시 클론닝하여 분석한 결과 이미 보고된 ${\alpha}1$-AT cDNA 서열과 일치함을 알 수 있었다. ${\alpha}1$-AT cDNA를 E. coli에서 발현시키기 위해 택 (tac) 프로모터에 의해 전사가 활성화되도록 발현벡터 pT-AT를 제조하였다. 플라스미드 pT-AT에 의해 형질전환된 E. coli JM109 균주에서 재조합 단백질의 발현을 유도한 결과 ${\alpha}1$-AT의 활성이 검증되었으며, 발현된 ${\alpha}1$-AT는 사람 피에서 정제한 ${\alpha}1$-AT와 그 면역학적 특성이 동일함을 알 수 있었다. 또한 같은 세포 추출액을 SDS-polyacrylamide 젤 전기영동법으로 분석한 결과 Coomassie Brilliant Blue로 단백질 밴드를 염색했을 때는 발현된 ${\alpha}1$-AT를 검증할 수 없었으나, ${\alpha}1$-AT에 대한 rabbit IgG 항체와 펄옥시다제가 연결된 anti-rabbit IgG goat 항체를 사용하여 웨스턴 블릿을 한 결과 ${\alpha}1$-AT 밴드가 기대했던 분자량 위치에서 확인되었다. 이와 같이 생산된 재조합 ${\alpha}1$-AT는 본래의 ${\alpha}1$-AT보다 분자량이 작은 것으로 나타났는데 이는 E. coli 세포내에서 발현된 ${\alpha}1$-AT는 glycosylation이 안된 형태의 것이기 때문일 것이다. Human ${\alpha}1$-AT cDNA was screened from human liver cDNA library, by use of oligonucleotide probes. DNA insert of 1.3 kbp from a positive clone was subcloned into pUC9 plasmid and was analyzed by restriction enzyme digestion and partial DNA sequencing. The obtained gene was identical to human ${\alpha}1$-AT cDNA reported previously. In order to express ${\alpha}1$-AT cDNA under regulation of tac promoter, expression vector pT-AT was constructed. E. coli JM109 cells transformed with pT-AT showed ${\alpha}1$-AT activity upon induction of recombinant protein synthesis. The recombinant ${\alpha}1$-AT was immunologically identical to authentic ${\alpha}1$-AT that was purified from human blood. When the recombinant cell lysates were analyzed on SDS-polyacrylamide gel electrophoresis, the expressed ${\alpha}1$-AT was not detected by staining with Coomassie Brilliant Blue. However Western blotting of the same gel, using rabbit IgG against ${\alpha}1$-AT and peroxidase-conjugated goat anti-rabbit IgG antibody, showed the existence of ${\alpha}1$-AT at the expected molecular weight region. Molecular weight of recombinant ${\alpha}1$-AT was slightly lower than that of authentic ${\alpha}1$-AT; which presumably was due to lack of glycosylation inside E. coli cells.

Pyridoxal - 5 ' - Phosphate 에 의한 Rhizobium trifolii Malonyl - CoA Synthetase 의 화학변형

이상철,김유삼 ( Sang Chul Lee,Yu Sam Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Pyridoxal-5`-phosphate inactivated malonyl-CoA synthetase from Rhizobium trifolii reversibly. The second-order rate constant, K_i was 2975 M^(-1) · min^(-1) at pH 7.0, 30℃. Inactivation of malonyl-CoA synthetase by PLP was reversed by dilution but not after reduction with NaBH₄. The spectra of malonyl-CoA synthetase, inactvated by PLP and reduced by NaBH₄, showed a characteristic Schiff base absorption change at 325 nm. ATP protected the enzyme from inactivation by PLP. These studies indicated that an essential amine is located at or near ATP binding region of the enzyme active site.

염화나트륨 농도가 느타리 자실체의 아미노산과 향기성분에 미치는 영향

이상철,편하영,박윤진,오태석,장명준,Lee, Sang-Chul,Pyeon, Ha-Young,Park, Youn-Jin,Oh, Tae-Seok,Jang, Myoung-Jun 한국버섯학회 2021 한국버섯학회지 Vol.19 No.4

본 연구는 염화나트륨에 의한 비생물적 스트레스가 생육과정 중의 느타리에게 미치는 영향을 자실체의 생육특성, 그리고 구성아미노산함량과 향기패턴 분석을 통해 확인하였다. 농도별로 염화나트륨을 처리한 톱밥배지에서 수확한 느타리 자실체의 생육특성을 조사한 결과, 처리구별 수량은 무처리구 대비 염화나트륨 0.5%처리구에서의 값은 비슷했으며, 염화나트륨 처리 농도가 1.0%에서 2.0%으로 증가하면서 수량이 확연히 감소하였다. 자실체의 구성아미노산함량 분석결과, 무처리구 대비 모든 염화나트륨 처리구에서 glutamic acid과 proline을 제외한 aspartate, threonine, serine, glycine, alanine, methionine, valine, isoleucine, leucine, phenylalanine, tyrosine, lysine, histidine, arginine의 14종 구성아미노산의 함량이 낮음을 확인하였다. 자실체의 향기패턴 분석결과, 염화나트륨 처리구에서 버섯고유의 향을 나타내는 octane compound로 추정되는 물질의 intensity가 감소하였음을 chromatography상에서 확인하였다. We investigated the effect of sodium chloride-associated abiotic stress on the development of Pleurotus ostreatus. We examined the growth characteristics of fruiting bodies, constituent amino acids, and fragrance pattern to determine the effect of culturing Pleurotus ostreatus on a sawdust substrate supplemented with sodium chloride in a dose-dependent manner. Pleurotus ostreatus fruiting bodies exhibited an increasing tendency towards augmented yields when grown in the presence of 0.5% sodium chloride as compared with that grown in the control group. However, increasing the supplementation of sodium chloride from 1.0 % to 2.0% resulted in significantly decreased yields of Pleurotus ostreatus fruiting bodies in these groups as compared with that in control groups. Further assessment revealed the presence of 14 types of amino acids in the fruiting bodies, including aspartate, threonine, serine, glycine, alanine, methionine, valine, isoleucine, leucine, phenylalanine, tyrosine, lysine, histidine, and arginine, at lower levels in all the sodium chloride-treated groups than in the control group; except for glutamic acid and proline. Similarly, fragrance pattern analysis of the Pleurotus ostreatus fruiting body by chromatography confirmed that the intensity of the substances presumed to be octane compounds, to which the unique flavor of mushrooms is attributed, was lower in all the sodium chloride-treated groups than in the control group.

Rhizobium trifolii Malonyl - CoA Synthetase 의 촉매반응에 관여하는 아미노산 Residue 의 화학변형

이상철,김유삼 ( Sang Chul Lee,Yu Sam Kim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Malonyl-CoA synthetase from Rhizobium trifolii was inactivated by 2,3-butanedione and diethylpyrocarbonate (DEP). In each case, inactivation followed pseudo first-order kinetics. The second-order rate constant for the inactivation by 2,3-butanedione was 17.4M^(-1)·min^(-1) at pH 8.0, 30℃. DEP rapidly inactivated the enzyme with the second-order rate constant, 780 M^(-1)·min^(-1) at pH 7.1, 30℃. The reaction order of inactivation with respect to reagents were 1.06 and 1.03 respectively. The substrate, ATP, afforded the protection against the inactivation of the enzyme caused by the reagent, respectively. These results indicate that arginine and histidyl residues essential for the enzyme activity are located at or near the ATP binding region of the active site.

In Vitro Translation of Glucose-6-Phosphate Dehydrogenase Isozyme mRNA and In Vivo Isotope Labeling of the Enzyme from Peking Duck

이상철,김유삼,Lee, Sang-Chul,Kim, Yu-Sam 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3

오리의 uropygial gland와 간에서 $poly(A)^+mRNA$를 분리하여 그들의 in vitro translation mixture로부터 pI 6.6인 glucose-6-phosphate dehydorgenase를 분리 하고 in vivo isotope labeling된 같은 효소를 분리하여 SDS polyacrylamide disc gel electrophoresis를 통하여 비교하였다. 그 결과 in vitro translation mixture로 부터 분리한 효소와 in vivo labeling된 효소는 그것이 uropygial gland의 것이거나 간의 것이거나 모두가 uropygial gland에서 직접 정제한 효소와 같은 크기임을 알 수 있었다. 이러한 사실은 uropygial gland에 존재하는 pI 6.6인 glucose-6-phosphate dehydrogenase가 갖는 효소적 특성은 소수의 아미노산의 차이에 기인하는 것으로 예측된다. One glucose-6-phosphate dehydrogenase with pI of 6.6 was isolated from in vitro translation mixture and was compared with in vivo isotopically labeled enzyme by SDS polyacrylamide gel electrophoresis. The molecular size of the enzymes, isolated by antibody prepared against the purified enzyme and protein A agarose, are very similar to each other. This result suggests that the specific catalytic properties of the enzyme from the uropygial gland may depend on the minor difference in amino acid sequence.

오리의 Urpygial Gland 에 있는 Glucose - 6 - phosphate Dehydrogenase 의 활성에 관여하는 Essential Cysteine 과 Arginine residues

이상철,김유삼 ( Sang Chul Lee,Yu Sam Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2

Glucose-6-phosphate dehydrogenase from the uropygial gland of duck was purified in an electrophoretically homogenous form and its essential amino acid for catalysis was investigated. Three -SH group directed reagents, p-chloromercuriphenylsulfonic acid, 5,5`-dithio-bis-(2-nitrobenzoic acid) and N-ethylmaleimide, inhibited the enzyme activity, and arginine directed reagent, phenylglyoxal, also inhibited the enzyme activity. The inhibition of the enzyme by these reagents was protected by addition of NADP+ whereas it was not affected by that of glucose-6-phosephate indicating that essential -SH and guanidino group are located on the NADP^+ binding region of active site. Mg^(++) promoted the inhibition of the enzyme by the -SH and guanidine .group directed reagents. But the promotion of the inhibition was considerably recovered in the presence of EDTA. This results suggested that divalent metal cation may affect the conformational change toward the susceptible one against modifying reagents.

광대싸리잎의 페놀성 화합물에 대한 화학적 연구

이상철,안병태,박웅양,이승호,노재섭,이경순,Lee, Sang-Chul,Ahn, Byung-Tae,Park, Woong-Yang,Lee, Seung-Ho,Ro, Jai-Seup,Lee, Kyong-Soon 한국생약학회 1994 생약학회지 Vol.25 No.2

A chemical examination of the aqueous acetone extract of the leaves of Securinega suffruticosa has led to the isolation of nine phenolic compounds. On the basis of chemical and spectroscopic evidences, the structures of these compounds were established to be gallic acid(1), corilagin(2), helioscopinin B(3), geraniin(4), bergenin (5), norbergenin(6), 11-O-galloyl norbergenin(7), gallocatechin(8) and rutin(9).

심리적 유인과 사이트 품질, 공동체의식이 온라인게임에 미치는 영향

이상철,김남희,문재영,서영호,Lee, Sang-Chul,Kim, Nam-Hee,Moon, Jae-Young,Suh, Young-Ho 한국경영정보학회 2003 Asia Pacific Journal of Information Systems Vol.13 No.4

The purpose of this research is to identify if psychological temptation, site quality and sense of community influence user's flow and addiction and to identify causalities among flow, addiction, customer satisfaction and customer loyalty in online industry. Many previous researches in the area of online games have been carried out about addiction by psychologist and about the development of related technologies by scientists. There are only a few studies about the customer satisfaction from the online business perspective. However, this research is different from the previous ones in the sense that both flow and addiction are considered in the study of the relationship between customer satisfaction/loyalty and flow/addiction in the area of online industry. The empirical results of high-order factor analysis indicate that six independent variables such as design, information, feedback, impulsiveness and motivation have converge three second-order variables such psychological temptation, site quality and sense of community. Consequently, site quality and sense of community have impacts on the flow, while on the other hand, psychological temptation has impacts on the addiction. Conclusively, customer satisfaction and loyalty are positively related not with the addiction but with the flow. Besides, customer loyalty is significantly influenced by the flow and the customer satisfaction. This indicates that companies in the online game industry have to develop a strategy for the flow which is more socially and ethically allowable than the addiction.