뱀 筋肉 Adenosine Triphosphate - Creatine Phosphotransferase에 關한 硏究

박충웅(Chung-ung Park) 한국식품영양과학회 1980 한국식품영양과학회지 Vol.9 No.1

A detailed procedure was described for the isolation of cratine kinase (ATP-Creatine phosphotransferase, E. C. 2. 7. 3. 2.) from the muscle of the snake Bungarus fasciatus. The original isolation procedure of Kuby et al. for the rabbit muscle enzyme has been modified and extended to include a chromatographic step.<br/> The properties of the enzyme have been investigated and kinetic constants for the reverse reactions determined as the followings:<br/> 1) A molecular weight of the enzyme was determined by gel filteration on Sephadex G-100 and by electrophoresis on SDS-polyacrylamide was 86,000.<br/> 2) Two reactive sulphydryl groups were detected with dithiobis nitrobenzoic acid (DTNB).<br/> 3) The nucleotide substrate specificity in the reverse reaction was determined as ADP*2'-dADP>GDP>XDP>UDP with magnesium as the activating metal ion.<br/> 4) The order of the metal specificity in the reverse reaction Mg>Mn>Ca~Co was determined with ADP as substrate.<br/> 5) A detailed kinetic analysis was carried out in the reverse direction with MgADP￣ as the nucleotide substrate. Initial velocity and product inhibition studies(MgATP^(2-) competitive with respect to MgADP- and noncompetitive with respect to N-phosphorycreatine^(2-) ; Creatine competitive with respect to N-phosphorylcreatine^(2-) and noncompetitive with respect to Mg ADP￣) indicated that the reaction obeyed a sequential mechanism of the rapid equilibrium random type.

Chlorella Protein의 營養價에 관한 硏究 (Ⅱ)

박충웅(Chung-Ung Park),황호관(Ho-Kwan Hwang) 한국식품영양과학회 1974 한국식품영양과학회지 Vol.3 No.1

It was reported that the digestion ratio of chlorella was low because it had a low metabolic rate in body.<br/> Generally, the thickness of a cell membrane of it is 200-250 Å, the weight of it is approximatly 13% of the total weight of a dry cell. And it is composed of protein, lipid, hemicellulose and ash etc.<br/> So, in order to elevate the digestion ratio of chlorella in body, we experimented the crude treatment methods of chlorella.<br/> The results obtained in this experiment are summarized as follows :<br/> 1. The digestion ratios calculated from ordinary N- balance method were 83.05% for 10% chl. (b) plus diastase group ; 81.25% for 10% chl. (b) plus amylase group, and 79.23% for 10% chl. (b), 58.55% for 10% chl. (a).<br/> 2. Biological values from this method were 80.25% for 10% chl. (b) plus diastase group, and 60% for 10% for chlorella(a).

카탈라제에 의한 프로파질알콜의 산화반응

곽진영,박충웅,이강민,Gwak, Jin Young,Park, Chung Ung,Lee, Kang Min 대한화학회 1999 대한화학회지 Vol.43 No.2

Vitreoscilla Catalase 의 분리 및 특성에 관한 연구

박기인,박충웅 ( Kie In Park,Chung Ung Park ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.6

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin and grows very well under the condition of low oxygen concentration. It also contain a bacterial catalase to be not correspond with another species on genus Beggiatoa. The primary function of Vitreoscilla catalase may be to remove hydrogen peroxide produced by ViWb oxidation. The molecular size of the catalase was estimated to be approximately 250,000 Da. The subunit structure of this enzyme may be A₂B₂ (A : MW 64,000 Da, B : MW 58,000 Da) but is not clear in the research reported here. Optimum pH is 7.0∼8.0 for catalase activity and Soret peak on absorption spectra of oxidized catalase is represented in 406 ㎚ and Soret peak of reduced form from sodium dithionite moved at 442 ㎚. Vitreoscilla catalase is unstable a high tempernture, and its Michealis constant, K_m was 0.016 M hydrogen peroxide. The turnover number of the enzyme was 25,000 mol. The 0.25 mM potassium cyanide was competitive inhibitor and the dissociation constant of the enzyme-inhibitor complex was 0.67 mM. N-terminal amino acid sequences of two subunits are determined for the probe synthesis using to the cloning of Vitreoscilla catalase gene.

피조개(Scapharca broughtonii)혈액 ferritin의 철분산화 기작에 관한 연구

손화숙,박충웅,김경숙,Sohn, Hwa-Suk,Park, Chung-Ung,Kim, Kyung-Suk 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.3

혈액내 철분의 농도가 비교적 높은(약 $4,000{\sim}5,000\;{\mu}gFe$/100ml) 피조개(Scaþharca broughtonii) 혈액에서 ferritin을 분리 정제하여 그 특성을 부분적으로 밝히고 ferritin core에 있어서 철분산화 기작을 연구하고자 단백질내 iron-core의 상태 및 성질을 조사하였으며 iron uptake시 초기 반응속도를 측정하였다. 이 단백질의 분자량은 500 kDa으로 산출되었고 subunit는 단일종으로 이루어져 있으며 size가 21 kDa이었다. 분리된 ferritin 분자내 함유된 철분은 평균 1,400~2,000 atoms이었다. Ferritin core의 상태는 전자현미경을 이용하여 ferrihydrite로 추정되었으며 M$\ddot{o}$ssbauer spectroscopy를 이용한 예비실험 결과 특이하게도 $T_{ord}$는 20 K 부근, $T_B$는 13 K보다 낮은 온도였다. 철분 산화반응에 있어서의 초기 반응속도는 Fe(II)의 농도에 비례하였고 단백질의 농도에 대해서도 비례함이 밝혀졌다. We have isolated ferritin from the blood of the marine bivalve mollusc Scapharca broughtonii in which blood iron levels were high (approx. $4,000{\sim}5,000\;{\mu}gFe$/100ml). The blood ferritin had a $M_r$ of ca. 500,000 with a single subunit type having a $M_r$ of ca. 21,000. The isolated ferritin contained 1,400~2,000 atoms Fe/molecule on average. The bivalve ferritin core was examined preliminarily using an electron microscope and its electron diffraction pattern implied characteristic lines of ferrihydrite. The preliminary M$\ddot{o}$ssbauer spectra of the S. broughtonii ferritin showed a striking difference. $T_{ord}$ was estimated ca. 20 K and $T_B$ was lower than 13 K. Ferroxidase activity of the bivalve apoferritin was assayed and characterized. Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). The initial rate of Fe(II) oxidation was dependent on Fe(II) concentration and a linear dependence of initial rate on protein concentration was observed.

피조개 ( Scapharca broughtonii ) 혈액 ferritin 의 철분산화 기작에 관한 연구

손화숙,박충웅,김경숙 ( Hwa Suk Sohn,Chung Ung Park,Kyung Suk Kim ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.3

We have isolated ferritin from the blood of the marine bivalve mollusc Scapharca broughtonii in which blood iron levels were high (approx. 4,000∼5,000 ㎍Fe/100 ml). The blood ferritin had a M_r of ca. 500,000 with a single subunit type having a M_r of ca. 21,000. The isolated ferritin contained 1,400∼2,000 atoms Fe/molecule on average. The bivalve ferritin core was examined preliminarily using an electron microscope and its electron diffraction pattern implied characteristic lines of ferrihydrite. The preliminary Mo¨ssbauer spectra of the S broughtonii ferritin showed a striking difference. T_(ord) was estimated ca. 20 K and T_B was lower than 13 K. Ferroxidase activity of the bivalve apoferritin was assayed and characterized. Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). The initial rate of Fe(II) oxidation was dependent on Fe(II) concentration and a linear dependence of initial rate on protein concentration was observed.

유기용매에 의한 EcoRI 제한효소의 특이성 변화

엄재영,박충웅,이강민,Um, Jae-Young,Park, Chung-Ung,Lee, Kag-Min 생화학분자생물학회 1994 한국생화학회지 Vol.27 No.5

DNA를 인식하여 절단하는 제한효소의 발견은 실험실에서 유전자를 연구, 조작할 수 있게되어 분자생물학 연구에 큰 발전을 가져왔다. 제한효소의 인식자리는 반응용액의 산도, 이온세기, 소수성, 유기용매, 효소의 양에 따라서 달라질 수 있다. 본 연구는 유전공학에서 가장 많이 이용되고 있으며 그의 3차 구조가 밝혀진 EcoRI 제한효소가 유기용매에 의한 특이성 변화를 연구하였다. 이 효소의 특이성은 에탄올, ethyleneglycol, DMSO와 같은 유기용매에 의하여 변화되며, 이 변화는 유기용매의 소수성(LogP)값과 밀접히 관계있다. EcoRI의 유기용매에 의한 특이성변화는 LogP값이 -2.0~0 사이에서 일어난다. Acetone, 2-methyl-propanol같은 그 효소를 쉽게 비활성화시키는 유기용매는 특이성을 변화에 영향을 주지못한다. 이러한 특이성 변화는 무질서하게 일어나지 않고 순서적으로 일어난다. 10% DMSO에서 EcoRI을 이용하여 pGEM3를 절단할 때 다음 절단자리는 TAATTC, GAGTTC순서로 절단된다. 이와 같은 제한효소의 반응조건을 바꾸면 고유의 절단자리가 아닌 다른자리를 절단할 수 있으며 이러한 기술은 유전공학에 이용될 수 있다. In molecular biology, type-II restriction endonuclease. which specifically cleave DNA at a limited number of sites, have been exploited as a means of characterizing DNA fragments, DNA mapping and of modifying DNA for genetic engineering. Recently, many type-II restriction endonucleases have been found to decrease their substrate specificity under modified conditions such as extreme pH, low ionic strength, high enzyme concentration, substitution of metallic cofactors, or addition of organic solvents. This study used restriction endonuclease EcoRI which are used most frequently in genetic engineering. We investigated their specificity change in buffer condition including various organic solvents. The specificity of cleavage of EcoRI is altered in the presence of hydrophobic reagents, such as ethanol, ethyleneglycol and DMSO. The enzyme recognition site was not changed randomly but by preferential order by increasing the concentration of organic solvent. When EcoRI reacted with substrate pGEM3 vector which have one canonical recognition site (GAATTC), EcoRI cleaved noncanonical TAATTC and GAGTTC subsequently in more than 10% DMSO solution. These changes of specificities depended on the hydrophobicity of organic solvent (LogP: partition coefficient). As a results, the recognition sequence site was changed in the presence of organic solvents whose LogP are -2.0~0. The specificities were not easily changed in enzyme inactivating organic solvent such as acetone, 2-methyl-2-propanol, 2-methyl-1-propanol. These results might show that restriction enzyme could be used to cleave at unusual site by changing the reaction condition.

고열충격반응에 영향을 미치는 초파리 hsp70 유전자의 DNA 염기배열

이영훈,전은순,박충웅 ( Young Hoon Lee,Eun Soon Jeon,Chung Ung Park ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

The DNA sequences required for heat shock regulated expression were examined in Drosophila melanogaster. Various hsp70-YP1 fusion constructs where D. melanogaster hsp70 gene and YP1 gene were used as a heat shock promoter and as a target gene, respectively, were transfected into D. melanogaster Schneider line 2 cells and their heat inducibility was determined by Northern analysis. The sequences containing two HSEs between -89 and -38 of the hsp70 were not sufficient to induce YP1 RNA upon heat shock whereas the sequences between -89 and +34 including the TATA box and the transcription start region of the hsp70 besides the HSEs were able to drive YP1 transcription during heat shock. The small deletion of the sequences near the transcription start region of the YP1 gene was also observed to repress the heat induced transcription of YPl RNA by the sequences between -89 and +34 of the hsp70.

고열충격반응에 영향을 미치는 초파리 hsp70 유전자의 DNA 염기배열

이영훈,전은순,박충웅,Lee, Young-Hoon,Jeon, Eun-Soon,Park, Chung-Ung 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

The DNA sequences required for heat shock regulated expression were examined in Drosophila melanogaster. Various hsp70-YP1 fusion constructs where D. melanogaster hsp70 gene and YPI gene were used as a heat shock promoter and as a target gene, respectively, were transfected into D. melanogaster Schneider line 2 cells and their heat inducibility was determined by Northern analysis. The sequences containing two HSEs between -89 and -38 of the hsp70 were not sufficient to induce YP1 RNA upon heat shock whereas the sequences between -89 and +34 including the TATA box and the transcription start region of the hsp70 besides the HSEs were able to drive YP1 transcription during heat shock. The small deletion of the sequences near the transcription start region of the YP1 gene was also observed to repress the heat induced transcription of YP1 RNA by the sequences between -89 and +34 of the hsp70. 초파리 Drosophila melalnogaster에서 고열충격 유발을 조절하는데 필요한 DNA 염기배열을 조사하였다. hsp 70 유전자를 고열충격 촉진자로, YP1 유전자를 표적유전자로 하여 hsp70-YP1 융합유전자를 제조하고 이들 융합유전자를 이용하여 초파리 Schneider line 2 세포를 형질변환시키고 Northern 분석을 함으로써 hsp70-YP1 융합유전자의 고열충격 유발성을 결정하였다. 두개의 HSE (heat shock element) 가 존재하는 hsp70 유전자의 -89에서 -38까지 염기배열만으로는 고열충격에 의해 YP1 RNA를 유발시킬 수 없었지만 두 개의 HSE 이외에 hsp70 유전자의 TATA 박스와 전사시작점을 포항하고 있는 -89에서 +34까지 염기배열은 고열충격에 의해 YP1 RNA를 유발시킬 수 있었다. 또한 YP1 유전자의 전사시 작정 근처의 염기배열이 조금 결실되었을 때 hsp70 유전자의 -89에서 +34까지 염기배열에 의한 YP1 RNA의 고열충격 유발이 억제되는 것이 관찰되었다.

전기자극에 의한 알콜 산화효소의 활성도와 안정도연구

이강민,김경숙,박충웅,Lee, Kang-Min,Kim, Kyung-Suk,Park, Chung-Ung 대한화학회 2004 대한화학회지 Vol.48 No.2

전기자극에서 알콜 산화효소의 활성도와 안정도를 연구하였다. 알콜 산화효소의 활성도와 안정도는 전기출력전압, 자극시간, 자극기간, 자극간격에 따라 달라진다. 전기자극에 의하여 비활성화 효소 활성도는 당, 중합체, 히드로젤과 같은 안정화 첨가제에 의하여 회복되었다. 이 효소에 전기자극을 40 V, 10분 주었을 때 완충용액에서는 활성도가 전혀 없었지만 10% 트레할로스 용액에서는 52%의 활성도를 유지하였다. 전기자극하에서 효소를 안정화시킬 수 있음은 효소를 생물공학과 의료공학에 널리 이용할 수 있는 가능성을 보여준다. We investigated the activity and stability of alcohol oxidase from Hansenula sp. under the electric stimulation. The activity and stability of alcohol oxidase depended on electric output voltage, stimulation time, pulse duration and pulse interval. This inactivation of the enzyme under electric stimulation could be recovered by stabilizing additives such as sugars, polymers and hydrogels. The enzyme activity retained about 52% in 10% trehalose solution under electric stimulation with 40 V and 10 min. The stabilizing of enzymes against electric stimulation showed a great potential use of enzymes in biotechnology and medical engineering fields.