콩과 식물에서 분리정제한 렉틴의 생물물리 화학적 연구(E-PHA 렉틴)

전경희,채영흠,서영아,정시련,JeuneChung, Kyung-Hee,Chae, Young-Heum,Suh, Young-Ah,Chung, See-Ryun 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.1

흰강남콩 (Phaseolus vulgaris)의 추출물을 DEAE Sephadex A-50과 hydroxyapatite column을 통과시켜 적혈구 및 립프구 응집효과를 갖는 EL-PHA를 얻었다. EL-PHA로부터 적혈구 응집효과만 가진 E-PHA을 분리하기 위해 EL-PHA를 4M urea로 처리한 다음 CM-Sephadex column을 통과시켜 림프구 응집력만 가진 L-PHA, 적혈구 응집력만 가진 EI-PHA, 적혈구 응집력과 약간의 림프구 응집력을 가진 ElI-PHA를 얻었다. EI-, EII-PHA의 생물물리화학적 성상을 구명코저 activity test, 전기영동, SDS-전기영동, 당류시험, 면역화학적 실험 등을 수행하였다. 전기영동에서 EL-PHA는 3개의 band를 나타내었고, EI-, EII-PHA는 1개의 뚜렷한 band를 나타내었으며, 분자량은 각각 130,000과 125,000 daltons으로 추정되었다. SDS-PAGE 결과 EI-PHA는 분자량이 약 34,000인 subunits 1종으로 나타났고, EII-PHA는 41,000과 30,000정도의 2종의 서로 다른 subunits로 나타났다. 당류에 의한 activity의 저해 효과는 0.3% D-mannose와 N-acetyl-D-galactosamine의 경우에 현저 하였고, D-galactose, L-fucose, N-acetylneuraminic acid, N-acetyl-D-glucosamine의 경우는 저해 효과가 없었다. 한편, EE-, EII-PHA 당류를 미량 함유한 당단백질임을 알 수 있었다. 면역 화학적인 연구의 하나로 immunodiffusion을 한 결과 EI-PHA는 antigen-antibody 반응을 잘 나타내 주었다. Salt fractionated extracts of Phaseolus vulgaris was purified successively through ion exchange chromatography on DEAE Sephadex A-50, hydroxyapatite column and EL-PHA was obtained. EL-PHA was treated with 4M urea just prior to the application into CM-Sephadex C-50 column. This column was eluted with 10 mM (pH 5.7), 20 mM (pH 6.6) and 30mM (pH 8.0) phosphate buffer, each containing 4M urea. In order to identify isolation of E-and L-PHA, erythroagglutination test with human blood A group and lymphoagglutinating test with mouse spleen lymphocytes were measured on each fraction and found three active fractions, L-PHA, having lymphoagglutinating activity only, EI-PHA, having high erythroagglutinating activity only and EII-PHA, having erythroagglutinating activity along with weak lymphoagglutinating activity. In order to characterize biophysicochemical properties of the EI-and EII-PHA, polyacrylamide disc gel electrophoresis, SDS gel electrophoresis, sugar inhibition test, carbohydrate test were performed. In polyacrylamide disc gel electrophoresis, EL-PHA was observed as three bands, EI-and EII-PHA were observed as clear single band with a very faint minor band. Molecular weight of EI-and EII-PHA were estimated at about 130,000 daltons and 125,000 daltons, respectively by PAGE. The results of SDS-PAGE shown that EI-PHA consists of four homogenous subunits having molecular weight of 34,000 per unit and EII-PHA consists of two different subunits with 41,000 arid 30,000 daltons. Erythroagglutination by EI-and EII-PHA was inhibited with 0.3% D-mannose and N-acetyl-D-galactosamine, but not with N-galactose, L-fucose, N-acetylneuraminic acid and N-acetyl-D-glucosamine. Sugar components of EI-and EII-PHA were recognized by anthrone test, but no spot was observed in paper chromatography. For immunochemical studies, antisera to EI-PHA were prepared by immunizing rabbit and EI-PHA formed precipitin bands against various antisera in Ouchterlony double immunodiffusion.

Lectins from Marine Shells: (VIII) Characterization of the NIC Lectin Isolated and Purified from Neptunea intersculpta

서영아,전경희,정시련,Suh, Young-Ah,JeuneChung, Kyung-Hee,Chung, See-Ryun 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.1

새로운 조각매물고둥렉틴인 NIC는 황화암모늄침전, 이온교환크로마토그래피 및 친화성크로마토그래피의 방법으로 분리, 정제되었다. 이는 전기영동상에서 한개의 주된 띠와 세개의 희미한 띠를 나타냈으며, specific activity는 82.16에서 85333.33 units/mg으로 증가되었다. Sephadex G-200 크로마토그래피와 SDS 전기영동결과, NIC는 분자량이 약 112,000으로 나타냈으며, 이는 분자량 약 14,000인 subunit의 octamer로 추정되었다. NIC는 2.5%의 당을 함유하는 당단백질이었으며, lactose, galactose 및 N-acetyl-D-galactosamine과 특이하게 결합하였다. 이의 아미노산조성은 산성아미노산의 함량이 많았으며, sulfur를 함유한 아마노산의 함량은 적었다. NIC, another Neptunea intersculpta lectin, was purified by salt fractionation, DEAE-cellulose 52 ion exchange chromatography and lactosyl-Sepharose 4B affinity chromatography. Through these purification procedures, specific activity of NIC was increased from 82.16 to 85333.33 units/mg. And on polyacrylamide gel electrophoresis, NIC exhibited one major and three minor bands. A molecular weight of NIC was calculated as 112,000 by Sephadex G-200 gel filtration and the subunit was determined as Mr 14,000 by sodium dodesyl sulfate polyacrylamide disc gel electrophoresis. Consequently, NIC is supposed to be an octamer composed of identical subunits of approximately Mr 14,000. NIC was a glycoprotein containing 2.5% of neutral sugar and had specificity for lactose, galactose and N-acetyl-D-galactosamine. NIC contained relatively high amounts of acidic amino acids, but the contents of sulfur containing amoino acids was very low.

한국산 식물 자원으로 부터 새로운 렉틴 성분의 분리 정제 (I) : 콩과 식물의 렉틴

정시련,전경희,Chung, See-Ryun,JeuneChung, Kyung-Hee 생화학분자생물학회 1981 한국생화학회지 Vol.14 No.3

In order to find new lectins from Korean natural products, 55 kinds of plants belonging to 31 families were screened by using several different type of blood cells. Phaseolus vulgaris C(W. K. B.) and Cercis chinenesis of Leguminosae contained strong hemagglutinating proteins. The crude lectins from W. K. B. were purified by DAEA Sephadex A-50 column chromatography. The 0.1 M peak from this procedure demonstrated acceptable criteria, e.g. yield, optical density and hemagglutinating activity and three bands were observed in polyacrylamide disc gel electrophoresis. A further purification was achieved by hydroxyapatite column chromatography and strong hemagglutinating activity was recovered in 0.1 M peak. It was elecrophoresed and found to migrate as a single band. The condition of purification for W. K. B. lectin was encourageable but did not brought agreeable satisfaction to the Cercis chinensis lectin. 한국산 식물 자원으로 부터 새로운 렉틴 성분을 개발하고 저 55종의 식물 자원을 대상으로 연구한 결과를 토대로 본 연구에서는 16종의 콩과식물 중 개발 가능성이 큰 Phaseolus vulgaris C와 Cercis chinensis로 부터 이 성분의 분리 정제를 시도하였다. DEAE-Sephadex A-50 column과 hydroxyapatite column으로 분리 정제를 하였고, polyacrylamide disc gel electrophoresis, UV spectrophotometry, 그리고 적혈구와의 agglutinating activity등으로 분리 정제된 렉틴을 비교 검토하였다. 본 연구에서 Phaseolus vulgaris 렉틴은 Cercis chinensis 렉틴에 비해 분리 정제 효과가 대단히 양호하였음을 관찰 하였다.

Studies on Lectins from Marine Shells (III) : Screening of Lectin-like Agglutinins from Marine Shells

Chung, See-Ryun,Kim, Jang-Hwan,Suh, Young-Ah,Jeunechung, Kyung-Hee The Pharmaceutical Society of Korea 1986 Archives of Pharmacal Research Vol.9 No.4

Forty species of marine shells were collected from Korean coasts and studied extensively for their lectin activities by using erythrocytes of human blooc A, AB, B. O group and rabbit blood. In total, 7 species contained lectines :Neptunea intersculpta, Omphalius nigerrimus and Scapharca subcrenata, blood group nonspecific; Saxidomus purpuratus, human blood A and AB group specific; Lepidozona coreana, Tegilarca granosa and Neptunea polycosta, rabbit blood specific.

해양패류로부터 렉틴성분 개발연구 VI : 조각매물고둥으로부터 새로운 렉틴의 분리 정제 및 특성

서영아,정시련,전경희,SuhChae, Young-Ah,Chung, Kyung-Hee,JeuneChung, Kyung-Hee 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.1

Netunea intersculpta로부터 새로운 렉틴을 0.15 M NaCl로 추출하여 $(NH_4)_2SO_4$로 침전분리시킨 후 DE 52, hydroxyapatite, chromatofocusing 등으로 정제 하였다 (NIB). NIB는 lactose, N-acetyl-D-galactosamine, D-galactose 특이성이고, 등전점은 pH 3.8이며, subunit는 분자량이 68,000, 66,000, 61,000의 세 종류로 추정 된다. 또한 $45^{\circ}C$까지의 온도에서는 렉틴활성이 안정하였다. NIB 렉틴은 산성아미노산의 함량이 높았으나, 유황을 함유한 아미노산은 확인되지 않았다. A new lectin(NIB) was obtained from the Neptunea intersculpta through the following procedure; 0.15 M NaCl extraction, ($(NH_4)_2SO_4$ salt fractionation, DEAE-cellulose 52, hydroxyapatite and chromatofocusing column chromatography. The hemagglutinating activity of the lectin was stable by $45^{\circ}C$ and was inhibited by lactose, N-acetyl-D-galactosamine and D-galactose. It's isoelectric point was pH 3.8 and it was composed of three kinds of subunits; 68,000, 66,000 and 61,000 daltons. The lectin contained relatively high contents of acidic amino acids, but the amount of sulfur cotaining amino acids was not estimated.