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doi:10.1016/j.peptides.2003.09.018 
Copyright © 2003 Elsevier Inc. All rights reserved.
Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme
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Pika Me
ko Brguljan a, b, Vito Turk a, Nina Cimerman a, c, Jo
e Brzin a, Igor Kri
aj a and Tatjana Popovi
, 
, a
a Department of Biochemistry and Molecular Biology, Jo
ef Stefan Institute, Jamova 39, Ljubljana 1000, Slovenia
b University Clinic for Respiratory Diseases and Allergy Golnik, 4204, Golnik, Slovenia
c Research and Development Division, Department of Biochemical Research and Drug Design, KRKA, d.d., Ljubljana 1000, Slovenia
Received 30 April 2003;
Abstract
Highly purified human brain cathepsin H (EC 3.4.22.16) was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1–6), dynorphin (1–13), α-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid
-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1–13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1–6), α-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [Δ1–10] variant, already known to be co-localized in astrocytes, since the Ki values for the inhibition are in the 10−10 M range.
Author Keywords: Author Keywords: Cathepsin H; Cystatin C; Truncated cystatin C; Neuropeptide; Bradykinin; Brain; Human
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Materials
- 2.2. Isolation of human brain cathepsin H
- 2.3. Determination of protein concentration
- 2.4. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and isoelectric focusing
- 2.5. N-terminal sequence analysis
- 2.6. Enzyme assays
- 2.7. Enzyme-linked immunosorbent assay (ELISA)
- 2.8. Purification of cysteine protease inhibitors
- 2.9. Determination of inhibition constants
- 2.10. Degradation of biologically active peptides
- 3. Results
- 3.1. Isolation and determination of purity of human brain cathepsin H
- 3.2. Inhibition of human brain cathepsin H with two variants of human cystatin C
- 3.3. Action of human brain cathepsin H on different biologically active peptides
- 4. Discussion
- Acknowledgements
- References
