DOI QR코드

DOI QR Code

Relationship Between Acrylamide Concentration and Enzymatic Activity in An Improved Single Fibrin Zymogram Gel System

  • Choi, Nack-Shick (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Byoung-Young (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Jin-Young (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yoon, Kab-Seog (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Han, Kyoung-Yoen (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Seung-Ho (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology)
  • 발행 : 2002.03.31

초록

Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDS-fibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10kDa to over 100kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

키워드

참고문헌

  1. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  2. Burg, B., Eijsink, V., Stulp, B. and Venema, G. (1990) Identification of autodigestion target sites in Bacillus subtilis neutral proteinase. Biochem. J. 272, 93-97. https://doi.org/10.1042/bj2720093
  3. Choi, N. S. and Kim, S. H. (1999) Application of fibrin zymography for determining the optimum culture time for protease activity. Biotechnol. Techniq. 13, 899-901. https://doi.org/10.1023/A:1008942611827
  4. Choi, N. S. and Kim, S. H. (2001) The effect of sodium chloride on the serine-type fibrinolytic enzymes and the thermostability of extracellular protease from Bacillus amyloliquefaciens DJ-4. J. Biochem. Mol. Biol. 34, 134-138.
  5. Choi, N. S., Yoon, K. S., Lee, J. Y., Han, K. Y., and Kim, S. H. (2001) Comparison of three substrate (casein, fibrin, and gelatin) in zymographic gel. J. Biochem. Mol. Biol. 34, 531-536.
  6. Hames, B. D. (1981) A practical approach; in Gel Electrophoresis, Hames, B. D. and Rickwood, D. (eds.), pp. 71-77, IRL Press, Oxford and Washington D.C.
  7. Heussen, C. and Dowdle, E. B. (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and co-polymerized substrates. Anal. Biochem. 102, 196-202. https://doi.org/10.1016/0003-2697(80)90338-3
  8. Jeong, E. J. and Han, O. S. (2001) Purification and characterization of Bacillus subtilis protoporphyrinogen oxidase and pre-equilibrium behavior during oxidation of protoporphyrinogen 9. J. Biochem. Mol. BioI. 34, 39-42.
  9. Kim, S. H. and Choi, N. S. (1999) Electrophoretic analysis of protease inhibitors in fibrin zymography. Anal. Biochem. 270, 179-181. https://doi.org/10.1006/abio.1999.4080
  10. Kim, S. H. and Choi, N. S. (2000) Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang. Biosci. Biotechnol. Biochem. 64, 1722-1725. https://doi.org/10.1271/bbb.64.1722
  11. 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. https://doi.org/10.1006/abio.1998.2816
  12. Kim, S. H., Choi, N. S., Lee, W. Y., Lee, J. W., and Kim, D. H. (1998) Isolation of Bacillus strains secreting fibrinolytic enzymes from Doen-Jang. Korean J. Microbiol. 34, 87-90.
  13. Kleiner, D. E. and Stetler-Stevenson, W. G. (1994) Quantitative zymography: detection of picogram quantities of gelatinases. Anal. Biochem. 218, 325-329. https://doi.org/10.1006/abio.1994.1186
  14. Kobayashi, T., Koike, K., Yoshimatsu, T., Higaki, N., Suzumatsu, A., Ozawa, T., Hatada, Y., and Ito, N. (1999) Purification and properties of a low-molecular weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus. Biosci. Biotechnol. Biochem. 63, 65-72. https://doi.org/10.1271/bbb.63.65
  15. Matsudaira, P. T. and Burgess, G. R. (1978) SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal. Biochem. 87, 386-396. https://doi.org/10.1016/0003-2697(78)90688-7
  16. Walker, J. M. (1984) Method in Molecular Biology; in Proteins, Walker, J. M. (ed.) Vol. 1, pp. 57-62, Humana Press, Totowa, New Jersey.

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