고상법에 의한 Bradykinin 유사물의 합성

최청,이재성,최무영,배만종,Choi, Cheong,Lee, Jae-Sung,Choi, Moo-Yung,Bae, Man-Jong 생화학분자생물학회 1981 한국생화학회지 Vol.14 No.4

고상법으로 개량된 기구에 의하여 bradykinin, $(Leu^1)$, $(Leu^2)$ 및 ($Ala^1$, $Ala^9$) bradykinin을 합성하였으며 이들 peptide의 정제 및 순도 측정에는 gel chromatography, paper chromatography, thin layer chromatography, paper electrophoresis, disc electrophoresis 및 아미노산 분석에 의거하였다. Endopeptidase인 ${\alpha}$-chymotrypsin과 trypsin, exopeptidase인 carboxypeptidase A와 leucine aminopeptidase를 사용하여 in vitro 상에서 이들 peptide의 분해 실험을 됐다. ${\alpha}$-Chymotrypsin과 carboxypeptidase는 peptide를 빠르게 분해하였으나 trypsin의 분해 작용은 느린 것으로 나타났다. Bradykinin, $(Leu^1)$과 ($Ala^1$, $Ala^9$) bradykinin에 는 N-말단에 모두 proline의 immino기와 결합되어 있기 때문에 leucine aminopeptidase의 작용을 받지 않았다. Synthesis of bradykinin, $(Leu^1)$, $(Leu^2)$ and ($Ala^1$, $Ala^9$) analogues of bradykinin by solid phase method using an improved reaction vessel was carried out. We employed gel chromatography, paper chromatography, thin layer chromatography, paper electrophoresis, disc electrophoresis and amino acid analysis for purification and for determining the purity of synthesized peptides. Bradykinin, $(Leu^1)$, $(Leu^2)$ and ($Ala^1$, $Ala^9$) analogues of bradykinin were incubated in vitro with endopeptidases (${\alpha}$-chymotrypsin, trypsin) and exopeptidase (carboxypeptidase A, leucine aminopeptidase) in girder to study the degradation patterns of the peptides. Eradykinin and $(Leu^1)$, $(Leu^2)$ and ($Ala^1$, $Ala^9$) analogues of bradykinin were rapidly degradated by ${\alpha}$-chymotrypsin and carboxypeptidase A, while the degradation by trypsin is slow. Bradykinin, $(Leu^1)$ and ($Ala^1$, $Ala^9$) analogues of bradykinin contain immino peptide bond from proline at N-terminal and therefore they were not attacked by leucine aminopeptidase.

한국재래간장으로 부터 분리한 Bacillus subtilis CCKS-111이 생성하는 Protease의 분리 및 정제

최청,김성,임성일,이희덕,이선호,손준호,최희진,김영활 ( Cheong Choi,Sung Kim,Seong Il Lim,Hee-Duck Lee,Seon Ho Lee,Jun Ho Son,Hee Jin Choi,Yeung hweal Kim ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.3

A protease was purified from Bacillus subtilis CCKS-111 by ammonium sulfate treatment, DEAE-cellulose ion-exchange chromatography, Sephadex G-100 gel filtration and high performance liquid chromatography (HPLC). The specific activity of the purified enzyme was 24.3 unit/㎎ protein and the purification fold of enzyme was 50.6. Molecular weight of the purified enzyme estimated about 28,000 by HPLC gel filtration. The amino acid residues of this enzyme were 251.3 except threonine, serine and glycine. This result was similar to Bacillus subtilis subtilisin DY. From the first N-terminal amino acid to the 32th amino acid, the amino acid sequence was estimated after RP-HPLC elution. N-terminal and the 32th amino acids were alanine and aspartic acid. Alanine, serine, glycine and arginine were four major acids in the enzyme.

한국 재래식 간장덧 발효시 대두 자숙 폐액 첨가가 젖산발효 촉진에 미치는 영향

최청,임무혁,최종동,정현채,김영호,이춘우,최광수,Choi, Cheong,Im, Moo-Hyeog,Choi, Jong-Dong,Chung, Hyun-Chae,Kim, Young-Ho,Lee, Choon-Woo,Choi, Kwang-Soo 한국응용생명화학회 1998 Applied Biological Chemistry (Appl Biol Chem) Vol.33 No.2

재래식 간장의 맛을 개선하기 위하여 대두 자숙액, 당 및 젖산균과 효모를 접종하고 자연조건에서 발효시키면서 젖산발효와 알코올발효 효과를 조사하였다. 젖산균과 효모 접종구에서 젖산의 생성량이 2.91%로 가장 높았고, 그 다음이 간장 덧에 당만을 첨가한 구에서 2.38%, 대두 자숙액 만을 첨가한 대조구에서 2.05%로 나타나 모든구의 간장에서 젖산발효의 효과가 있었으나, 알코올은 모든 처리구에서 소량 검출되어 젖산균에 의해 생성된 초산 때문에 효모의 생육이 저해되어서 알코올발효 효과는 없는 것으로 나타났다. 기호도에서도 젖산균과 효모 접종구와 당 첨가구가 우수하였다. In order to evaluate the effects of addition of soybean boiling waste liquor (SBWL) and sugar and inoculation of the lactic acid bacteria and yeast starter culture in Korean traditional kanjang mash, three types of kanjang were prepared in a clay pot of 100 l volume and compared the characteristics of lactic acid fermentation. The mashing compositions of the types of kanjang were as follows: (1) control treatment mash was prepared with meju : 20% salt solution (1:4) and SBWL, (2) kanjang mash with 3.5% added sugar to the control type mash and (3) kanjang mash with 3.5% added sugar and inoculation of the lactic acid bacteria and yeast starter culture 35 days after mashing to the control type mash. (1), (2) and (3) of kanjang mash were found to be effective in increasing the lactic acid content and improving the organoleptic characteristics of kanjang. But the effect of yeast starter culture was not clear because osmophilic yeasts were inhibited by metabolite(acetic acid) produced by lactic acid bacteria. The lactic acid content of (1), (2) and (3) kanjang was 2.05, 2.38 and 2.91% respectively in 90 day-matured kanjang.

에지추적에 의한 영상 분할 및 부호화

최청,이상미,김남철,손현,Choi, Cheong,Lee, Sang-Mi,Kim, Nam-Chul,Son, Hyon 대한전자공학회 1989 전자공학회논문지 Vol. No.

A new simple edge-based segmentation method composed of edge tracing, region filling, and post processing is proposed. Solving so called the small gap problem common to most of edge-based methods, this method segments images so completely as to be suitable for image coding. Experimental results show that our methods has much less (1/3) computation time than Perkins' one, and its reconstructed images is good on visual perception. 에지추적, 영역채색 및 후처리 과정으로 구성된 간단한 에지기반 분할법을 새로이 제안하였다. 이 방법은 대부분 에지기반법에서 나타나는 소극문제를 해결함으로써, 영상부호화에 적용할 수 있을 정도로 완전하게 영상을 분할한다. 실험결과, 이 방법은 Perkins방법보다 3배정도 빠르고, 그 재생영상도 인식면에서 우수한 것으로 나타났다.

Pseudomonas sp. SJ-320로부터 알칼리성 단백질 가수분해효소의 생산과 특성

최청,김영활,천성숙,Choi, Cheong,Chun, Sung-Soak,Kim, Yung-Hawr 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.6

알칼리성 단백질 가수분해효소의 생산능력이 강한 Pseudomoηas sp. SJ-320을 토양으로부터 분리하였으며 효소생산의 최적 배양조건은 배지에 1% casein, 0.2% ammonium chloride, 0.2% $NaH_{2}PO_{4}$, 1% fructose를 혼합첨가하여 $20^{\circ}C$, pH 10.0에서 6일간 배양하였을 때 효소생산이 최대로 나타났다. 효소의 최적 작용 pH와 온도는 pH 8.0, $50^{\circ}C$ 였으며, pH 7.5-8.5의 범위와 $50^{\circ}C$ 이하에서 안정하였다. 급속이온 중 $Mn^{2+}$, $Mg^{2+}$과 $Ca^{2+}$ 등에 의하여 활성이 증대되었으나 $Hg^{2+}$, $Cu^{2+}$ 등에 의하여 효소활성이 저해되었다. 2,4-dinitrophenol, ${\varepsilon}-aminocaproic$ acid 처리에 의해 활성이 저해되지 않았으나 ethylenediaminetetraacetic acid와 p-chloromercuribenzoic acid에 의해 활성이 저해되어 효소분자 중 sulfhydryl기가 활성에 어느 정도 관여하는 metallo enzyme으로 추정되였다. 이 효소는 sodium dodecyl sulfate을 제외한 여러 가지 계면활성제에 대해서 강한 저항성을 가지고 있었다. An alkaline protease producing microorganism was isolated from soil and identified as Pseudomonas sp. SJ-320. The optimum cultivation condition of Pseudomonas sp. SJ-320 for the production of alkaline protease was as follow; 1.0% casein, 0.2% ammonium chloride, 0.2% $NaH_{2}PO_{4}$, 1.0% fructose, $20^{\circ}C$, pH 10.0 and 140 h. The optimum pH and temperature for the enzyme activity were pH 8.0 and $50^{\circ}C$, respectively. The enzyme was relatively stable at pH 7.5-8.5 and at temperature below $50^{\circ}C$. The activity of the partially purified enzyme was inhibited by $Hg^{2+}$ and $Cu^{2+}$ whereas $Mn^{2+}$, $Mg^{2+}$ and $Ca^{2+}$ gave rather activating effects on the enzyme activity. ${\varepsilon}-Aminocaproic$ acid and 2,4-dinitrophenol did not inhibit the enzyme, but p-chloromercuribenzoic acid and ethylendiaminetetraacetic acid inhibited the enzyme activity, indicating that reactive sulfhydryl group and metal ion group are required for the enzyme activity. The enzyme was resistant to various detergents, except for sodium dodecyl sulfate.

Synthesis of Angiotensin II Analogues by Solid Phase Method

최청,Choi, Cheong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.1

고상법으로 개량된 반응용기를 이용하여 angiotensin II 유사물인 ($Ala^1$), ($Ala^2$) 및 ($Ala^3$) angiotensin II를 합성하였으며 그 수득률은 62.2에서 68.0%이었다. Pressor activity는 alanine이 N- 말단에 가까울수록 감사하였으며 그 최대치는 80.5%이었다. 이들 펩티드의 정제 및 순도결정은 gel filtraction, paper chromatography, thin layer chromatography, 전기영동, 녹는점 측정 및 아마노산 분석기에 의거하였다. Synthesis of ($Ala^1$), ($Ala^2$) and ($Ala^3$) analopgues of angiotensin II by solid phase method using an improved reaction vessel was carried out. The yield average 62.2-68.0%. Homogeneity and purity of the three analogues synthesized peptides were established with paper chromatography, paper electrophoresis, amino acid analysis and melting point apparatus. The pressor activity was gradually increased with the increase in the chain length from the N-terminal to Ala residue angiotensin II, however the maximum value was within 80.5%.

돼지의 Adrenal Medulla에서 Leucine 및 Methionine Enkephalin 분리

최청,배만종,Choi, Cheong,Bae, Man-Jong 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.1

High pressure liquid chromatography를 이용하여 돼지의 adrenal medulla로 부터 leucine 및 methionine enkephalin을 분리 한 후 아미노산 조성 및 아미노산 배열을 규명한 실험 결과를 얻었다. 1. 이들 아편성 peptide의 순수 분리에는 gel chromatography, CM-cellulose chromatography, thin layer chromatography, HPLC를 이용하였다. 2. peptide인 leucin enkephalin과 아미노산 조성의 몰 비율은 Tyr : 1.00, Gly : 1.96, Phe : 1.03, Leu: 1.03, Leu: 1.00. Methionine enkephaline은 Tyr : 1.00, Gly : 1.98, Phe : 1.02, Met: 1.01 이었다. 3. pentapeptide인 leucine 및 methionine enkephalin을 medulla로부터 추출 분리하였으며, 아미노산 배열은 H-Tyr-Gly-Gly-Phe-Leu-OH; leucine enkephalin, H-Tyr-Gly-Gly-Phe-Leu-OH였다. Leucine and methionine enkephalin was isolated from porcine adernal medulla by use of high pressure liquid chromatography (HPLC), and the amino acid composition and sequence of isolated peptides were elucidated. 1. We empolyed gel chromatography, CM-Cellulose chromatography, thin layer chromatography, amino acid analysis and HPLC for the purificatition of opioid peptides. 2. The molar fraction ratio of amino acids of pentapeptides, leucine enkephalin, is shown to be Tyr: 1.00, Gly: 1.96, Phe: 1.03, leu: 1.00, and that of methionine enkephalin is shown to be Tyr: 1.00, Gly: 1.98, Phe: 1.02, Met: 1.01. 3. Leucine and methionine enkephalin has been isolated from acid exttracts of porcine adrenal medulla and found to have the amino acids sequence H-Tyr-Gly-Phe-Leu-OH and H-Tyr-Gly-Gly-Phe-Met-OH.

Methionine Enkephalin 유사물의 합성에 관한 연구 (I)

최청,윤상홍,안봉전,Choi, Cheong,Yoon, Sang-Hong,An, Boug-Jeun 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.4

개량된 반응기구를 사용하여 고상법으로 methionine enkephalin 및 ($Phe^1$), (D-$Ala^1$), (D-$Ala^2$) enkephalin을 합성하였으며 이들의 수득율은 55~65%였다. Coupling은 dicyclohexycarbodimide에 의하여 이루어 졌으며 HBr으로 Cleavage한 각 펩티드는 gel filteration 및 ion-exchange chromatography에 의해 분리하였고 이들의 순도 결정은 thin layer chromatography, paper chromatography, paper electrophoresis, melting point 및 아미노분석에 의거 하였다. Methionine enkephalin 및 ($Phe^1$), (D-$Ala^1$), (D-$Ala^2$) enkephalin을 in-vitro 상에서 endopeptidase ($\alpha$-chymotrypsin, trypsin) 과 exopeptidase (carboxypeptidase, leucineaminopeptidase)를 사용하여 이들의 분해 실험을 했다. $\alpha$-chymotrypsin, carboxypeptidase 및 leucine aminopeptidase는 peptide를 빠르게 분해 시켰으나 trypsin의 작용은 받지 않았다. Synthesis of methionine enkephalin and ($phe^1$), (D-$ala^1$), (D-$ala^2$) analogues of methionine enkephalin was carried by solid phase method. The yield ranged from 55 to 65% owing to an improved reaction vessel. Coupling was performed by dicyclohexylcarbodiimide. After cleavage with dried HBr, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin layer chromatography, paper electrophoresis, melting point and amino acid analysis. Methionine enkephalin and ($phe^1$), (D-$ala^1$), (D-$ala^2$) analogues of methione enkephalin were incubated in vitro with endopeptidase ($\alpha$-chymotrypsin, trypsin) and exopeptidase (carboxypeptidase A, leucine aminopeptidase) in order to study the degradation patterns of peptides. Methionine enkephalin and ($phe^1$), (D-$ala^1$), (D-$ala^2$) analogues of methionine enkephalin were rapidly degradated by $\alpha$-chymotrypsin, carboxypeptidase A and leucine aminopeptidase and therefore they were not attacked by trypsin.

식품착색료가 ${\alpha}$-Chymotrypsin 작용에 미치는 영향

최청,김상옥,Choi Cheong,Kim, Sang Ok 대한화학회 1977 대한화학회지 Vol.21 No.6

단백질 분해효소인 ${\alpha}$-chymotrypsin이 식품발색제의 존재하에 어떻게 oligopeptide에 작용하여 분해하는가를 알기 위하여 본 실험을 실시하였다. 1. Oligopeptide인 Asp-Arg-Val-Tyr-Ile-His-Pro-D-Ala(8-D-Ala${\cdot}$angiotensin II)의 융점은 210 ~ $212^{\circ}C$, 분자식은 $C_{44}H_{67}N_{13}O_{12}{\cdot}2CH_3COOH{\cdot}H_2O$, 분자량은 970.08이었다. 2. 산으로 가수분해 하였을 때 몰 비율은 Asp : 1.01, Arg : 1.03, Val : 1.00, Tyr :40.94, Ile : 1.00, His : 1.05, Pro : 1.04, D-Ala : 1.03이였다. 3. (8-D-Ala)angiotensin II의 oligopeptide에 ${\alpha}$-Chymotrypsin의 작용은 Tyr-Ile 결합에만 분해작용을 하였다. 4. 식품착색제의 첨가는 paper chromatogram 방법에 의해서 추정할때 oligopeptide, (8-D-Ala) angiotensin II에 대한 ${\alpha}$-Chymotrypsin의 저해작용에 아무런 영향을 끼치지 않았음을 볼 수 있다. This study was carried out to understand the activity of ${\alpha}$-chymotrypsin, a proteolytic enzyme, to a oligopeptide in the presence of various coloring food additives. 1. The melting point of synthetic oligopeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-D-Ala, ((8-D-Ala) angiotensin Ⅱ) was 210∼$212^{\circ}C$. Chemical formula and molecular weight were $C_{44}H_{67}N_{13}O_{12}{\cdot}2CH_3COOH{\cdot}H_2O$ and 970.08, respectively. 2.The amino acid rations by acid hydrolysis were Asp : 1.01, Arg : 1.03, Val : 1.00, Tyr :40.94, Ile : 1.00, His : 1.05, Pro : 1.04, D-Ala : 1.03. 3. ${\alpha}$-Chymotrypsin cleaved the oligopeptide bond between tyrosine and isoleucine (Tyr-Ile). 4. The addition of food coloring additives as determined by paper chromatogram, did not influence the inhibitory activity of ${\alpha}$-chymotrypsin on oligopeptide, (8-D-Ala) angiotensin II.

고상법에 의한 Bradykinin 유사물의 합성 (II)

최청,이재성,배만종,윤상홍,Choi, Cheong,Lee, Jae-Sung,Bae, Man-Jong,Yoon, Sang-Hong 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.4

Bradykinin(BK)의 receptor $B_1$ 상에서 nonapeptide의 구조 및 활성에 대한 연구 목적으로 bradykinin유도체 ($Leu^1$, $Leu^2$)BK, ($Leu^2$, $Leu^3$)BK, ($Ala^7$, $Ala^9$)BK 및 ($Ala^8$, $Ala^9$)BK을 고상법으로 합성하였다. coupling은 N,N'-dicyclohexylcarbodiimide로 행하였으며 HBr응액으로 cleavage한 후 peptides는 Sephadex G-25와 carboxymethyl cellulose칼럼 크로마토 그라피로 정제하였다. 이들 peptides의 순도측청은 paper chromatography, thin layer chromatography, paper electrophoresis, slab gel electrophoresis. melting point 및 아미노산 분석에 따랐다. endopeptidase인 ${\alpha}$-chymotrypsin과 trypsin, exopeptidase인 carboxypeptidase A와 leucine aminopeptidase를 사용하여 이들 peptides의 분해실험을 하였다. 즉 carboxypeptidase A와 ${\alpha}$-chymotrypsin은 이들을 빠르게 분해하였고 trypsin은 느리게 분해 하였으나, ($Ala^7$, $Ala^9$) 및 ($Ala_8$, $Ala^9$)BK은 N-말단에 모두 proline의 imino와 결합되었기 때문에 leucine aminopeptidase의 작응을 받지 않았고 ($Leu^1$, $Leu^2$)BK은 1번 위치에 arginine과 같은 방향족 아미노산이 치환되지 않았으므로 trypsin의 작응을 받지 않았다. We have synthesized, by solid phase method, a series of 4 analogues of the nonapeptide, bradykinin(BK) in order to conduct a structure-activity study of these peptides on the newly discovered bradykinin $B_1$ receptor. Coupling were performed by dicyclohexylcarbodimide. After cleavage with liquid HBr, peptides were purified by gel fitration on Sephadex G-25 and ion-exchange chromatography on carboxymethyl cellulose. The purity of the nonapeptide was then checked by paper chromatography, thin layer chromatography, paper electrophresis slab gel electrophoresis, melting point and amino acid analysis. ($Leu^1$, $Leu^2$), ($Leu^2$, $Leu^3$), ($Leu^7$, $Leu^9$) and ($Leu^8$, $Leu^9$) analogues of bradykinin were incubated in vitro with endopeptidase ($\alpha$-chymotrypsin, trypsin) and exopeptidase (carboxypeptidase A, leucine aminopeptidase) in order to study the degradation patterns of peptides. ($Leu^1$, $Leu^2$), ($Leu^2$, $Leu^3$), ($Leu^7$, $Leu^9$) and ($Leu^8$, $Leu^9$) analogues of bradykinin were rapidly degraded by $\alpha$-chymotrypsin and carboxypeptidase A, while the degradation by trypsin was slow. But ($Ala^7$, $Ala^9$) BK and ($Ala^8$, $Ala^9$)BK contain imino peptide bond from proline at N-terminal and therefore they were not attacked by leucine aminopeptidase.