Korean Journal of Medicinal Crop Science (한국약용작물학회지)

The Korean Society of Medicinal Crop Science (한국약용작물학회)
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Fibrinolytic Enzyme Activity of Extract from Camellia japonica L.

동백나무 추출물의 혈전용해 효소활성

  • Lim, Chae-Young (Biotechnology Industrialization Center, Dongshin University) ;
  • Lee, Sook-Young (Research Center for Proteineous Materials, Chosun University) ;
  • Pyo, Byeong-Sik (Department of Oriental Medicine Material, Dongshin University) ;
  • Kim, Sun-Min (Department of Oriental Medicine Material, Dongshin University)
  • 임채영 (동신대학교 생물자원산업화지원센터) ;
  • 이숙영 (조선대학교 단백질소재연구센터) ;
  • 표병식 (동신대학교 한약재산업학과) ;
  • 김선민 (동신대학교 한약재산업학과)
  • Published : 2006.08.30
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Abstract

The fibrinolytic activities of soluble proteins extracted from young leaves of Camellia japonica L. were studied. Fibrinolvity activity of extract from partitions of C. japonica L. showed 1.6-2.0 times higher than plasmin used as positive control. The fibrinolytic enzyme was confirmed directly from young leaves of C. japonica L. by a fibrin Plate and fibrin zymography. The protein was composed of a single polypeptide and its apparent molecular weight was found to be 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the fibrinolytic activity were pH 5.5 and $30^{\circ}C$, respectively. Also, the fibrinolytic activity was clearly inhibited by PMSF and TLCK, suggesting that it is a member of the trypsin-like serine protease. All these results suggest the protease is a fibrinolytic enzyme belong to a family of trypsin-like serine protease.

미생물 및 동물에 비해 식물에서는 혈전용해효소에 대한 연구가 매우 부족한 실정이며, 기존의 혈전용해효소가 가지는 혈전에 대한 비특이적, 부작용, 고가 등의 단점을 해결할 수 있는 새로운 혈전용해효소의 개발을 위하여 동백의 종자, 종피, 유엽 그리고 성엽으로부터 추출된 수용성 단백질의 혈전용해활성을 조사하였다. 각각의 동백부위로부터 추출된 조효소 용액은 기존 혈전 용해효소인 plasmin과 양성 대조군으로 하여 비교하여 fibrin agarose plate로 확인한 결과 fibrin clot을 효과적으로 분해하였다. 그 중 단백질 분해효소의 활성이 다른 부위보다 20-33배로 높았던 동백종자와 종피의 수용성 추출물의 혈전용해활성은 양성 대조군인 plasmin과 비교하여 1.6-2.0배의 높은 활성을 나타내었다. 전체 수용성 단백질은 30-80%황산암모늄을 이용하여 농축하였으며 혈전용해효소는 fibrin zymography를 수행하여 확인하였다. SDS-PAGE에 의하여 동백유엽의 혈전용해효소 분자량을 측정한 결과 45 kDa으로 단일 polypeptide임을 확인하였으며, 각종 pretense 저해제에 의한 영향을 조사한 결과 PMSF,그리고 TLCK에 강력하게 저해되는 것으로 보아 동백유엽의 혈전용해효소는 trypsin과 유사한 serine protease의 하나로 생각되었다. 그러나 EDTA와 DTT처리에 의해서는 효소활성의 저해가 두드러지게 나타나지 않고 오히려 증진된 양상을 확인할 수 있었다. 또한, 효소 활성에 미치는 pH 및 온도의 효과는 약간의 산성쪽에 가까운 pH 5.5와 $30^{\circ}C$에서는 최적의 활성을 나타내었으며, $45^{\circ}C$ 이상의 온도에서는 효소활성이 급격히 감소하였다. 이상의 모든 결과로 볼 때 동백유엽의 혈전용해효소는 trypsin과 유사한 serine protease에 속하는 혈전용해효소임을 확인할 수 있었다.

Keywords

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