[논문]A Method for Direct Application of Human Plasmin on a Dithiothreitol-containing Agarose Stacking Gel System

Choi, Nack-Shick; Chung, Dong-Min; Yoon, Kab-Seog; Maeng, Pil-Jae; Kim, Seung-Ho

doi:10.5483/bmbrep.2005.38.6.763

[국내논문] A Method for Direct Application of Human Plasmin on a Dithiothreitol-containing Agarose Stacking Gel System 원문보기

Journal of biochemistry and molecular biology = 한국생화학회지, v.38 no.6, 2005년, pp.763 - 765

Choi, Nack-Shick (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) , Chung, Dong-Min (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) , Yoon, Kab-Seog (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology) , Maeng, Pil-Jae (Department of Microbiology, Chungnam National University) , Kim, Seung-Ho (Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology)

Abstract ▼ AI-Helper

A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1%) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.

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제안 방법

5 mm thickness), and electrophoresis was performed in the second dimension. In this study, a new DTT copolymerizsed ASG system was developed to identify human plasmin. Using this method, the primary structure of human plasmin could be established under acidic conditions.
This study developed an improved and simplified procedure for identifying disulfide-linked enzyme using a DTT copolymerized ASG system. The ASG method has two distinct advantages over the diagonal electrophoresis method (Brennan et al.

대상 데이터

Reagents The human fibrinogen, thrombin, plasmin, Triton X100, and DTT were purchased from Sigma (St. Louis, USA). The other chemicals were of analytical grade.
3 and Table 1). This protein was found to consist of three fragments, 45 kDa (spot 1), 23 kDa (sopt 2), and 13 kDa (spot 3). In addition, its primary structure was also determined based on the report by Wu et al.

이론/모형

The membrane was then stained with Coomassie blue. The stained portion was excised and used directly for N-terminal sequencing using a Gas-phase protein sequencer (model Procise 491, ABI, USA) according to the automated Edman degradation method.

참고문헌 (9)

Brennan, J. P., Wait, R., Begum, S., Bell, J. R., Dunn, M. J. and Eaton, P. (2004) Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis. J. Biol. Chem. 279, 41352-41360

상세보기
Choi, N. S., Hahm, J. H., Maeng, P. J. and Kim, S. H. (2005) Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, ${\beta}$ - mercaptoethanol, DTT, SDS, Triton X-100, and urea. J. Biochem. Mol. Biol. 38, 177-181

원문보기 상세보기
Choi, N. S. and Kim, S. H. (2000) Two fibrin zymography methods for analysis of plasminogen activators on gels. Anal. Biochem. 281, 236-238

상세보기
Kim, S. H. and Choi, N. S. (1999) Electrophoretic analysis of protease inhibitors in fibrin zymography. Anal. Biochem. 270, 179-181

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Kim, S. H., Choi, N. S. and Lee, W. Y. (1998) Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Anal. Biochem. 263, 115-116

상세보기
Schaller, J., Moser, P. W., Dannegger-Muller, G. A. K., Rosselet, S. J., Kampfer, U. and Rickli, E. E. (1985) Complete amino acid sequence of bovine plasminogen: comparison with human plasminogen. Eur. J. Biochem. 149, 267-278

상세보기
Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoridemembranes. J. Biol. Chem. 262, 10035-10038
Wu, H. L., Shi, G. Y. and Bender, M. L. (1987a) Preparation and purification of microplasmin. Proc. Natl. Acad. Sci. USA 84, 8292-8295
Wu, H. L., Shi, G. Y., Wohl, R. C. and Bender, M. L. (1987b) Structure and formation of microplasmin. Proc. Natl. Acad. Sci. USA 84, 8793-8795

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