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Comparison of Kinetic Properties of Native and Recombinant Human Cathepsin D

  • Paula E. Scarborough
  • Gary R. Richo
  • John Kay
  • Gregory E. Conner
  • Ben M. Dunn
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 306)

Abstract

The aspartic proteinase cathepsin D has been shown to function primarily in the degradation of proteins in the lysosomes of most higher eukaryotic cells and, recently, has been evaluated as a prognostic factor in node-negative breast cancer. Synthesized as a preproenzyme and cleaved cotranslationally to proenzyme, procathepsin D is then further processed to the single-chain and finally two-chain active forms of cathepsin D which reside in the lysosome. The rapidity of the initial cleavage of procathepsin D makes it difficult to isolate the zymogen form. In order that the processing1 and active site requirements of cathepsin D may be examined, human fibroblast procathepsin D has been expressed in E. coli, refolded from solubilized inclusion bodies, and purified using a pepstatin affinity column. To confirm that the recombinant enzyme is an acceptable model for studying substrate specificity and the processing mechanism in vitro, kinetic properties of recombinant cathepsin D were compared to those of native cathepsin D isolated from human placenta and spleen. Kinetic parameters were determined for a series of synthetic chromogenic peptide substrates derived from a parent substrate with the following sequence.

Keywords

Aspartic Proteinase Sodium Formate Human Cathepsin High Eukaryotic Cell Solubilized Inclusion Body 
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.

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Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Paula E. Scarborough
    • 1
  • Gary R. Richo
    • 2
  • John Kay
    • 3
  • Gregory E. Conner
    • 2
  • Ben M. Dunn
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of FloridaGainesvilleUSA
  2. 2.Department of Anatomy and Cell BiologyUniversity of MiamiMiamiUSA
  3. 3.Department of BiochemistryUniversity of Wales College of CardiffCardiffUK

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