Biologia Plantarum

, 26:99 | Cite as

Detection and evaluation of serine proteinase by affinity chromatography on immobilized-aprotinin inRicinus communia

  • Tsuneo Watanabe
  • Noriaki Kondo
  • Kazutaka Kano
Original Papers


Neutral proteinase was found in the leaves ofRicinus communie as assayed with α-casein and H-D-Val-Leu-Lys-pNA as substrates. The enzyme is maximally active at pH around 7.4. A selective adsorbent for serine proteinase was prepared by attaching aprotinin to aminoalkyl-porous glass.

When partially purified leaf proteinase was passed through a column containing this adsorbent, the proteinase activity present was bound to the porous glass. The proteinase eluted at IM NaCl was inhibited by aprotinin, leupeptin, DFP, phenylmethylsulfonyl fluoride (PMSF) and serine proteinase inhibitor fromR, communis leaves, whereas pepstatin, EDTA, EGTA, and DTT had no effect on the enzyme. This inhibition profile suggests the leaf proteinase is a neutral proteinase, such as a serine proteinase.


Serine Proteinase Proteinase Activity Affinity Chromatography Plasmin Porous Glass 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations used








polyethylene glycol-6000


  1. Baumgartner, B., Chrispeels, M. J.: Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans. - Plant Physiol.58: 1–6, 1976.PubMedGoogle Scholar
  2. Bradford, M. M.: A rapid and sensitive method for the quantities of protein utilizing the principle of protein-dye binding. -Anal. Biochem.72: 248–254, 1976.PubMedCrossRefGoogle Scholar
  3. Castellino, F. J., Powell, J. R.: Human plasminogen. - In:Lorand, L. (ed.): Methods in Enzymology. Vol. 80. Pp. 365–378. Academic Press, New York 1981.Google Scholar
  4. Chrispeels, M. J., Boulter, D.: Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase. - Plant Physiol.55: 1031–1037, 1975.PubMedGoogle Scholar
  5. Dalling, M. T., Boland, G., Wilson, J. H.: Relation between acid proteinase activity and redistribution of nitrogen during grain development in wheat. - Aust. J. Plant Physiol.3: 721–730, 1976.CrossRefGoogle Scholar
  6. Drivdahl, R. H., Thimann, K. V.: The proteases of senescing oat leaves, II. Reaction to substrate and inhibitors. Purification and general properties. - Plant Physiol.61: 501–505, 1978.PubMedGoogle Scholar
  7. Feller, U. K., Soong, T. T., Hageman, R. H.: Leaf proteolytic activities and senescence during grain development of field-grown corn (Zea mays L.). - Plant Physiol.59: 290–294, 1977.PubMedGoogle Scholar
  8. Frith, G. J. T., Swinden, L. B., Dalling, M. J.: Proteolytic enzymes in green wheat leaves. II. Purification by affinity chromatography and some properties of some proteinases with acid pH optima. - Plant Cell Physiol.19: 1029–1041, 1978.Google Scholar
  9. Hobday, S. M., Thurman, D. A., Barber, D. J.: Proteolytic and trypsin-inhibitory activities in extracts of germinatingPisum sativum seeds. - Phytochemistry12: 1041–1046, 1973.CrossRefGoogle Scholar
  10. Holzer, H., Betz, H., Ebner, E.: Intracellular proteinases in microorganisms. - In:Horecker, B. L., Stadtmann, E. R. (ed.): Current Topics in Cellular Regulation. Vol. 9. Pp. 103–155. Academic Press, New York 1975.Google Scholar
  11. Larsson, P. C., Mosbach, K.: Preparation of NAD(H)-polymer matrix. Showing coenzyme function of the bound pyridine nucleotide. - Biotechnol. Bioeng.13: 393 - 398, 1971.PubMedCrossRefGoogle Scholar
  12. Martin, C., Thimann, K. V.: The role of protein synthesis in the senescence of leaves, I. The formation of protease. - Plant Physiol.49: 64–71, 1972.PubMedGoogle Scholar
  13. Miller, B. L., Huffaker, R. C.: Partial purification and characterization of endoproteinases from senescing barley leaves. - Plant Physiol.68: 930–936, 1981.PubMedGoogle Scholar
  14. Pusziai, A.: Metabolism of trypsin-inhibitory proteins in the germinating seeds of kidney bean. - Planta107 : 121–129, 1972.CrossRefGoogle Scholar
  15. Royer, A., Miege, M. N., Grange, A. J., Miege, J., Mascherpha, J. M.: Inhibiteurs antitrypsine et activités protéolytiques des albumines de graine deVigna unguiculata. - Planta119:1–16, 1974.CrossRefGoogle Scholar
  16. Ryan, C. A.: Proteolytic enzymes and their inhibitors in plants. - Annu. Rev. Plant Physiol.24:173–196, 1973.CrossRefGoogle Scholar
  17. Salmia, M. A., Mikola, J. J.: Inhibitors of endogenous proteinases in the seeds of Scots pine,Pinus silvestris. - Physiol. Plant.48: 126–130, 1980.CrossRefGoogle Scholar
  18. Shain, Y., Mayer, A. M.: Activation of enzymes during germination trypsin-like enzyme in lettuce. -Phytochemistry7: 1490–1498, 1968.CrossRefGoogle Scholar
  19. Watanabe, T.: Mechanisms of change of the structure of protein and degradation in a cellular milieu by hydrogen peroxide and protease. - Medic. Biol.104: 103–105, 1982.Google Scholar
  20. Watanabe, T., Kano, K.: Purification and characterization of plasmin inhibitor fromSpinacia oleracea. -Blood Ves.13: 314–317, 1982.Google Scholar
  21. Watanabe, T., Kondo, N.: The change in leaf protease and protease inhibitor activities after supplying various chemicals. - Biol. Plant.25: 100–109, 1983.Google Scholar
  22. Wittenbach, V. A.: Breakdown of ribulose bisphosphate carboxylase and changes in proteolytic activity during dark induced senescence of wheat seedlings. - Plant Physiol.62: 604 to 608, 1978.CrossRefGoogle Scholar

Copyright information

© Academia 1984

Authors and Affiliations

  • Tsuneo Watanabe
    • 1
  • Noriaki Kondo
    • 1
  • Kazutaka Kano
    • 2
  1. 1.Laboratory of Biochemistry and Physiology, Division of Environmental BiologyNational Institute for Environmental StudiesIbarakiJapan
  2. 2.Department of Physiological Chemistry and Nutrition Faculty of MedicineUniversity of TokyoTokyoJapan

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