Anti-Putrescine 항체의 제조 및 이를 이용한 Putrescine의 정량

이승관,조영동,Lee, Seung-Gwan,Cho, Young-Dong 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Heterobifunctional cross-linker인 GMBS를 사용하여 mercaptosuccinylation된 BSA에 putrescine을 결합시켜 토끼에 주사하여 putrescine에 대한 항체를 얻었다. Ouchterlony double immunodiffusion에서 Anti-putrescine serum은 BSA-putrescine과는 침강대를 형성하였으나 BSA과는 침강대를 형성하지 않았다. Anti-putrescine serum은 cadaverine과는 5.3%, spermme과는 3.5% 그리고 spermidine과는 2.3%의 교차 반응성을 보여 주었으나 1,3-diaminopropane과, lysine에 대해서는 교차 반응성이 없었다. Nitrocellulose에 결합시킨 항체와 $[1,4-^{14}C]$putrescine을 이용하여 putrescine에 대한 radioimmunoassay을 개발하였다. 이 측정법에 의한 최저 측정농도는 100 pmol/tube이였다. 대두에서 HPLC에 의한 putrescine치와 TLC에 의한 putrescine치 각각을 RIA에 의한 치에 대하여 상관관계를 조사한 결과 모두 좋은 상관을 보여주었다. 또한 이 측정방법은 putrescine의 유도체로 만들지 않아도 재현성, 특이성 및 정확성이 높았으며 putrescine을 빠르고 간단하게 측정할 수 있었다. An antibody directed toward putrescine has been produced in rabbits by immunization with a putrescine-protein conjugate which was prepared by a procedure of coupling putrescine to mercaptosuccinylated bovine serum albumin using $N-({\gamma}-maleimidobutyryloxy)succinimide$ as a coupling agent. In the analysis of ouchterloney double immunodiffusion, anti-putrescine serum produced precipitation line with BSA-putrescine, but not with BSA. Anti-putrescine serum showed 5.3% crossreaction with cadaverine, 3.5% with spermine, and 2.3% with sper-midine but not crossreactions with 1,3-diaminopropane and lysine. An radioimmunoassay for putrescine has been developed utilizing anti-putrescine serum bound to nitrocellulose and $[1,4-^{14}C]$putrescine. The lower limit of detection by this assay was 100 pmol/tube. In the correlation studies of putrescine determined by this methods (RIA, HPLC, TLC method), RIA showed good correlation with other two methods. This RIA method was turned out to be reproducible, specific, accurate, rapid and simple for determination of putrescine without making derivative of putrescine.

대두 하배축과 Cotyledon으로부터 정제된 Betainealdehyde dehydrogenase의 생화학적 특성 및 면역학적 성질에 관한 연구

이승관,조영동,Lee, Seung-Gwan,Cho, Young-Dong 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.8

대두 cotyledon으로부터 정제된 betainealdehyde dehydrogenase(BADH)의 분자량은 120,000 dalton이었으며 60,000 dalton의 homodimer로 대두 하배축의 효소와 같게 나타났다. 두 효소 모두의 betainealdehyde에 대한 $K_m$값은 큰 차이를 보이지 않았다. 하배축의 BADH는 $NAD^+$에 대해 특이성이 있었으나 cotyledon의 BADH는 $NAD^+$에 대해 특이성을 보이지 않았다. 또한 cotyledon의 BADH는 polyamine들(spermidine, sprmine, putrescine, agmatine, cadaverine)에 의해 저해되지 않았으나 대두 하배축의 BADH는 polyamine들에 의해 저해되었다. 대두하배축의 BADH에 대한 항체에 의해 두 효소 모두 단일 침강대를 형성하였으며 두 조직의 BADH 사이에는 부분적 동일성이 나타났다. Western blotting 분석에서는 두 조직의 효소 모두 같은 크기의 단백질에서 면역 반응성을 보여주었다. Immunogold staining을 실시한 후 그 표본을 전자 현미경으로 관찰한 결과 하배축과 cotyledon 모두에서 vacuole내에 존재하는 단백질체에서 gold particle들이 나타났다. The molecular weight of the native enzyme was 120,000, while the subunit molecular weight was 60,000, indicating a homodimer. The $K_m$ values of the axes enzyme and cotyledonous enzyme for betaine aldehyde were almost similar. BADH from axes had high specificity toward $NAD^+$ whereas BADH from cotyledons did not have. Amines including polyamines were turned out to be innocuous for cotyledonous BADH but inhibit the enzyme activity of axes. BADH from two different tissues formed precipitation line against anti-BADH antiserum. There was shown partial identity between two BADH from soybean cotyledons and axes. In the western blotting analysis, immunoreactive band of enzymes from two different soybean tissues was shown on the same location. Through the immunocytochemical localization, immunegold particles were shown in the protein bodies of vacuole.

대두 하배축과 Cotyledon 으로부터 정제된 Betainealdehyde dehydrogenase 의 생화학적 특성 및 면역학적 성질에 관한 연구

이승관,조영동 ( Seung Gwan Lee,Young Dong Cho ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.8

The molecular weight of the native enzyme was 120,000, while the subunit molecular weight was 60,000, indicating a homodimer. The K_m values of the axes enzyme and cotyledonous enzyme for betainealdehyde were almost similar. BADH from axes had high specificity toward NAD+ whereas BADH from cotyledons did not have. Amines including polyamines were turned out to be innocuous for cotyledonous BADH but inhibit the enzyme activity of axes. BADH from two different tissues formed precipitation line against anti-BADH antiserum. There was shown partial identity between two BADH from soybean cotyledons and axes, In the western blotting analysis, immunoreactive band of enzymes from two different soybean tissues was shown on the same location. Through the immunocytochemical localization, immunegold particles were shown in the protein bodies of vacuole.

대두에서 단백질에 결합된 Polymaine 에 관한 연구

이승관,조영동 ( Seung Gwan Lee,Young Dong Cho ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Quantitative analysis of different forms of polyamines was accomplished in Glycine max, seed soaked in water for 24 h. The content of bound polyamine was turned out to be higher than those of free and conjugated polyamines. Acid hydrolysates of glycinins purified from soybean cotyledons showed that 1 g of the glycinin contained 14.2∼16.0 μ㏖ of put, 18.2∼ 22.3 μ㏖ of spermidine, and 4.4∼7.3 μ㏖ of spermine, respectively. Through immunocytochemical localization of putrescine it was found in vacuolar protein bodies, which contain glycinin.

Anti - Putrescine 항체의 제조 및 이를 이용한 Putrescine 의 정량

이승관,조영동 ( Seung Gwan Lee,Young Dong Cho ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

An antibody directed toward putrescine has been produced in rabbits by immunization with a putrescine-protein conjugate which was prepared by a procedure of coupling putrescine to mercaptosuccinylated bovine serum albumin using N-(γ-maleimidobutyryloxy)succinimide as a coupling agent. In the analysis of ouchterloney double immunodiffusion, anti-putrescine serum produced precipitation line with BSA-putrescine, but not with BSA. Anti-putrescine serum showed 5.3% crossreaction with cadaverine, 3.5% with spermine, and 2.3% with sper-midine but not crossreactions with 1,3-diaminopropane and lysine. An radioimmunoassay for putrescine has been developed utilizing anti-putrescine serum bound to nitrocellulose and [1,4-^(14)C]putrescine. The lower limit of detection by this assay was 100 p㏖/tube. In the correlation studies of putrescine determined by this methods (RIA, HPLC, TLC method), RIA showed good correlation with other two methods. This RIA method was turned out to be reproducible, specific, accurate, rapid and simple for determination of putrescine without making derivative of putrescine.

대두에서 단백질에 결합된 Polyamine에 관한 연구

이승관,조영동,Lee, Seung-Gwan,Cho, Young-Dong 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Glycine max를 24시간 물에 담그면서 여러 형태의 polyamine을 관찰한 결과 결합형의 polyamine들이 conjugated form이나 자유형에 비해 많이 존재하였다. 주 저장단백질인 glycinin을 정제한 후 이들을 가수분해하여 polyamine을 분석한 결과 putrescine, spermidine, spermine을 각각 glycinin 1g당 $14.0{\sim}16.2{\mu}mol$, $18.8{\sim}22.4{\mu}mol$, $4.4{\sim}7.3{\mu}mol$ 함유하고 있었다. Putrescine에 대한 항체로 immunocytochemical localization을 수행한 후 전자현미경으로 관찰하였더니 대부분의 면역학적인 반응성은 단백질체에서 나타났다. Quantitative analysis of different forms of polyamines was accomplished in Glycine max, seed soaked in water for 24 h. The content of bound polyamine was turned out to be higher than those of free and conjugated polyamines. Acid hydrolysates of glycinins purified from soybean cotyledons showed that 1g of the glycinin contained$14.2{\sim}16.0{\mu}mol$ of put, $18.2{\sim}22.3{\mu}mol$ of spermidine, and $4.4{\sim}7.3{\mu}mol$ of spermine, respectively. Through immunocytochemical localization of putrescine it was found in vacuolar protein bodies, which contain glycinin.

흰쥐 수뇌의 퓨트리신 분포에 관한 전자현미경을 이용한 면역세포화학적 연구

이종은,박경아,이승관,조영동 대한해부학회 1991 Anatomy & Cell Biology Vol.24 No.4