해녀콩의 발아와 생장시 Canavanine 의 이용과 Canavanase 의 활성에 대하여

권영명(Young Myung Kwon),정흥채(Hung Chae Chung),고석찬(Suck Chan Koh),홍영남(Young Nam Hong) 한국식물학회 1986 Journal of Plant Biology Vol.29 No.2

Canavanine content of the cotyledons of Canavalia lineata decreased gradually during germination and growth of seedlings but continued to increase in roots and leaves. After abscission of cotyledons, canavanine content of leaves depleted competely. The activity of canavanase could be detected in leaves and roots, but not in cotyledons. High arginase activity was observed in the cotyledons of seeds at the early imbibition period. During the growth of seedlings, cotyledonary canavanine appeared to be transported to the growing poprtions of the seedlings where it could be utilized through nitrogen metabolic pathways. In crude cell-free extracts of leaves, maximum activities of canavanase or arginase appeared in 30mM Tris-HCl buffer (pH 9.0) or 30mM NaHCO_3 buffer (pH 10.0), respectively. The activities of these two enzymes differed from each other when treated with Co^2+ or Mn^2+. These results support the idea that canavanase and arginase might be different enzymes.

해녀콩의 유식물 (幼植物) 자엽내 BAρNAase 의 성질

고석찬(Suck Chan Koh),허인옥(In Ok Heo),권영명(Young Myung Kwon) 한국식물학회 1994 Journal of Plant Biology Vol.37 No.2

N-α-benzoyl-DL-arginine ρ-nitroanilide hydrolase (BAρNAase) was partially purified and characterized from the cotyledons of Canavalia lineata. The overall purification and final recovery were 77.5-fold and 7%, respectively. The apparent mol wt of the native enzyme was estimated as 200 kD from the gel filtration and this enzyme was revealed as one of the gelatin-hydrolyzed proteases by activity staining with gelatin-containing gel. The optimum pH for the hydrolysis of N-α-benzoyl-DL-arginine ρ-nitroanilide (BAρNAase) was about 9.5. This enzyme catalyzed the hydolysis of BAρNA with Vmax of 15.5 unit/mg and Km value of 1.6 mM which were different from them of bovine-pancreatic trypsin. The catalytic activity of this enzyme was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) but insensitive to aprotinin, indicating that it was a serine proteinase but not a trypsin-like one. And this enzyme was slightly stimulated by Ca^2+ and mg^2+ but remarkably inhibited by Mn^2+, HG^2+ and Zn^2+.

해녀콩 ( Canavalia Lineata ) 자엽에서 Canavalin 의 일부 특성과 변화

고석찬,황인두,권영명 ( Suck Chan Koh,In Doo Hwang,Young Myung Kwon ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6

The properties and fate of canava1in were investigated in the cotyledons during germination and growth of Canavalia lineata. The molecular weights of canavalin and its subunit were determined by Sephacryl S-300 column chromatography and SDS-PAGE, leading to the inference that canavalin is consistent with the trimer structure consisting of 49 kD subunit. The canavalin subunit had an apparent isoelectric point of approximately 5.5 based on denaturing IEF and was hydrolyzed by trypsin into two fragments of 27 kD and 25 kD representing cleavage at one vulnerable point in the polypeptide chain for trypsin. The canavalin subunit and its cleavaged products react with the antiserum to the canava1in subunit and the canava1in subunit and 25 kD polypeptide degraded during germination and growth of seedlings, but 24 kD polypeptide remained until the 8th day after sowing.

해녀콩(Canavalia lineata) 자엽에서 Canavalin의 일부 특성과 변화

고석찬,황인두,권영명,Koh, Suck-Chan,Hwang, In-Doo,Kwon, Young- Myung 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6

해녀콩(Canavalia lineata)의 자엽에서 canavalin을 분리하여 그 특성을 알아보고 발아와 유식물 생장시 자엽에서 canavalin의 변화를 조사하였다. Sephacryl S-300 컬럼 크로마토그래피와 SDS-전기영동으로 canavalin과 그 subunit의 분자량이 각각 165kD와 49kD로 canavalin은 3량체임을 알았다. 그리고 등전점 전기영동으로 canavalin subunit의 등전점이 5.5이고 trypsin에 의하여 27kD와 25kD의 분해산물을 생성함을 알았다. 한편, 건조한 종자에는 canavalin과 그 subunit 그리고 25kD와 24kD의 작은 폴리펩티드가 존재하나 canavalin subunit는 점차 분해되고 25kD의 폴리펩티드는 4~6일 이후에 모두 분해되지만 24kD의 폴리펩티드는 8일까지도 비교적 다량존재하였다. The properties and fate of canavalin were investigated in the cotyledons during germination and growth of Canavalia lineata. The molecular weights of canavalin and its subunit were determined by Sephacryl S-300 column chromatography and SDS-PAGE, leading to the inference that canavalin is consistent with the trimer structure consisting of 49kD subunit. The canavalin subunit had an apparent isoelectric point of approximately 5.5 based on denaturing IEF and was hydrolyzed by trypsin into two fragments of 27kD and 25kD representing cleavage at one vulnerable point in the polypeptide chain for trypsin. The canavalin subunit and its cleavaged products react with the antiserum to the canavalin subunit and the canavalin subunit and 25kD polypeptide degraded during germination and growth of seedlings, but 24kD polypeptide remained until the 8th day sowing.

해녀콩(Canavalia lineata) 유식물 자엽에서 Canavalin 분해효소의 분리 및 특성

고석찬,고정군,황인두,권영명,Koh, Suck-Chan,Koh, Jung-Goon,Hwang, In-Doo,Kwon, Young-Myung 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.6

해녀콩(Canavalia lineata) 자엽에서 canavalin 분해활성을 갖는 endopeptidase를 분리하여 합성기절에 대한 특이성과 protease 억제제의 효과를 조사하였다. Canavalin 분해효소는 DEAE-Sephacel 컬럼에 붙지 않는 분획을 Sephacryl S-300 컬럼을 통과시켜 분리하였으며, 분자량이 각각 200 kD(protease I)와 27 kD(protease II)인 2종으로 밝혀졌다. 이들 protease I과 II는 공통적으로 49kD의 canavalin 단위체에 작용하여 25kD과 24kD의 폴리펩티드를 생성하였으며, EDTA와 phenanthroline에 의하여 활성이 크게 억제되어 metalloproteinase 로 판단되었다. 그러나, protease I은 Cbz-ala-ONp, Cbz-leu-ONp 등의 지방족 사슬을 갖는 기질을, protease II는 Cbz-asn-ONp, Cbz-gln-ONp 등의 아미드기를 가진 기질을 잘 분해하였다. Two canavalin-hydrolyzing proteases (proteases I and II) from the cotyledons of Canavalia lineata were isolated and characterized. These proteases were isolated by Sephacryl S-300 column chromatography from fractions excluded from DEAE-Sephacel column and elucidated to hydrolyze the 49 kD canavalin subunit into two fragments of 25 kD and 24 kD. The approximate molecular weights of proteases I and II were estimated as 200 kD and 27 kD, respectively. The proteases I and II were inhibited by EDTA and phenanthroline, and so proteases I and II seemed to be metalloproteinases. The protease I readily hydrolyzed synthetic ester between ${\rho}-nitrophenol$ and amino acid having aliphatic side chain, and the protease II readily hydrolyzed esters with amino acids having amide group.

해녀콩 ( Canavalia lineata ) 유식물 자엽에서 Canavalin 분해효소의 분리 및 특성

고석찬,고정군,황인두,권영명 ( Suck Chan Koh,Jung Goon Koh,In Doo Hwang,Young Myung Kwon ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.6

Two canava1in-hydrolyzing proteases (proteases Ⅰ and Ⅱ) from the cotyledons of Canavalia lineata were isolated and characterized. These proteases were isolated by Sephacryl S-300 column chromatography from fractions excluded from DEAE-Sephacel column and elucidated to hydrolyze the 49 kD canava1in subunit into two fragments of 25 kD and 24 kD. The approximate molecular weights of proteases Ⅰ and Ⅱ were estimated as 200 kD and 27 kD, respectively. The proteases ⅹ and Ⅱ were inhibited by EDTA and phenanthroline, and so proteases Ⅰ and Ⅱ seemed to be metalloproteinases. The protease Ⅰ readily hydrolyzed synthetic ester between ρ-nitrophenol and amino acid having aliphatic side chain, and the protease Ⅱ readily hydrolyzed esters with amino acids having amide group.

Canavanine 에 의한 보리 무배부 (無胚部) 종자의 Amylase 활성과 단백질 함량의 변화

전방욱(Bang Ook Jun),고석찬(Suck Chan Koh),권영명(Young Myung Kwon) 한국식물학회 1983 Journal of Plant Biology Vol.26 No.4

L-canavanine was added to GA_3 treated barley seeds, and induced amylase activity, soluble protein content, and arginine content were measured. Canavanine, added at the beginning of the incubation period, inhibited amylase activity and protein accumulation. Amylase activity decreased markedly by addition of canavanine at 6 hr after incubation, where soluble protein content was not affected. The addition of canavanine after 12 hr incubation did not show serious inhibitory effect on the amylasa activity and protein accumulation. GA_3 incubation caused decrement in arginine content per ㎎ protein, but it was somewhat recovered by canavanine treatment. The longer the time between GA_3 and canavanine addition was, the less the recovery ratio was. Arginine content in the α-amylase fraction (ammonium sulfate 20∼50% saturation) was lower than in 0∼20% fraction, but higher than in 50∼80% fraction. These results and control experiments, using cordycepin and cycloheximide, support the idea that canavanine might incorporate into protein.

해녀콩 ( Canavalia lineata ) 잎 칼루스의 유도와 유리 아미노산의 분석

황인두(In Doo Hwang),고석찬(Suck Chan Koh),권영명(Young Myung Kwon) 한국식물학회 1991 Journal of Plant Biology Vol.34 No.3

The callus of Canavalia lineata was induced from leaf tissues in MS medium supplemented with 10^-5 M kinetin and 10^-6 M IAA and was subcultured in Miller`s medium supplemented with 10^-5 M BAP and 10^-6 M 2,4-D. When free amino acids of callus were analysed by HPLC, canavanine was not detected in the callus cultured either in the dark or light. But exogenously supplied canavanine was accumulated or consumned in the callus of Canavalia lineata.

해녀콩(Canavalia lineata)자엽에서 Canavalin의 일부 특성과 변화

권영명,황인두,고석찬 濟州大學校 基礎科學硏究所 1992 基礎科學硏究 Vol.5 No.1

해녀콩(Canavalia lineata)의 자엽에서 canavalin을 분리하여 그 특성을 알아보고 발아와 유식물 생장시 자엽에서 canavalin의 변화를 조사하였다. Sephacryl S-300 컬럼 크로마토그래피와 SDS-전기영동으로 canavalin과 그 subunit의 분자량이 각각 165kD와 49kD로 canavalin은 3량체임을 알았다. 그리고 등전점 전기영동으로 canavalin subunit의 등전점이 5.5이고 trypsin에 의하여 27kD와 25kD의 분해산물을 생성함을 알았다. 한편, 건조한 종자에는 canavalin과 그 subunit 그리고 25kD와 24kD의 작은 폴리펩티드가 존재하나 canavalin subunit는 점차 분해되고 25kD의 폴리펩티드는 4∼6일 이후에 모두 분해되지만 24kD의 폴리펩티드는 8일까지도 비교적 다량존재하였다. The properties and fate of canavalin were investigated in the cotyledons during germination and growth of Canavalia lineata. The molecular weights of canavalin and its subunit were determined by Sephacryl S-300 column chromatography and SDS-PAGE, leading to the inference that canavalin is consistent with the trimer structure consisting of 49kD subunit. The canavalin subunit had an apparent isoelectric point of approximately 5.5 based on denaturing IEF and was hydrolyzed by trypsin into two fragments of 27kD and 25kD representing cleavage at one vulnerable point in the polypeptide chain for trypsin. The canavalin subunit and its cleavaged products react with the antiserum to the canavalin subunit and the canavalin subunit and 25kD polypeptide degraded during germination and growth of seedlings, but 24kD polypeptide remained until the 8th day sowing.