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A new selective substrate for cathepsin E based on the cleavage site sequence of α2-macroglobulin

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Published/Copyright: July 5, 2005
Biological Chemistry
From the journal Volume 386 Issue 3

Abstract

Cathepsin E is an intracellular aspartic proteinase of the pepsin family predominantly expressed in cells of the immune system and believed to contribute to homeostasis by participating in host defense mechanisms. Studies on its enzymatic properties, however, have been limited by a lack of sensitive and selective substrates. For a better understanding of the importance of this enzyme in vivo, we designed and synthesized a highly sensitive peptide substrate for cathepsin E based on the sequence of the specific cleavage site of α2-macroglobulin. The substrate constructed, MOCAc-Gly-Ser-Pro-Ala-Phe-Leu-Ala-Lys(Dnp)-D-Arg-NH2[where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], derived from the cleavage site sequence of human α2-macroglobulin, was the most sensitive and selective for cathepsin E, with kcat/Km values of 8–11 μM-1 S-1, whereas it was resistant to hydrolysis by the analogous aspartic proteinases cathepsin D and pepsin, as well as the lysosomal cysteine proteinases cathepsins B, L, and H. The assay allows the detection of a few fmol of cathepsin E, even in the presence of plasma and cell lysate, and gives accurate results over a wide enzyme concentration range. This substrate might represent a useful tool for monitoring and accurately quantifying cathepsin E, even in crude enzyme preparations.

Keywords: aspartic proteinase; α2-macroglobulin; cathepsin E; selective substrate
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Published Online: 2005-07-05
Published in Print: 2005-03-01

©2004 by Walter de Gruyter Berlin New York

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https://doi.org/10.1515/BC.2005.036
Keywords for this article
aspartic proteinase; α2-macroglobulin; cathepsin E; selective substrate

Articles in the same Issue

  1. Signaling in Biochemical Pharmacology and Toxicology
  2. Oncogenic Ras in tumour progression and metastasis
  3. Phosphoinositide 3-kinase signaling in the cellular response to oxidative stress
  4. Doxorubicin induces EGF receptor-dependent downregulation of gap junctional intercellular communication in rat liver epithelial cells
  5. TGFβ-induced focal complex formation in epithelial cells is mediated by activated ERK and JNK MAP kinases and is independent of Smad4
  6. On the mechanism of alkylphosphocholine (APC)-induced apoptosis in tumour cells
  7. Self-organization versus Watchmaker: stochastic dynamics of cellular organization
  8. Varicella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex
  9. Trehalose and 6-aminohexanoic acid stabilize and renature glucose-6-phosphate dehydrogenase inactivated by glycation and by guanidinium hydrochloride
  10. Quercetin metabolism in vital and apoptotic human leukaemia cells
  11. One of the Ca2+ binding sites of recoverin exclusively controls interaction with rhodopsin kinase
  12. Enzymatic profiling of human kallikrein 14 using phage-display substrate technology
  13. A new selective substrate for cathepsin E based on the cleavage site sequence of α2-macroglobulin
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