The localization of cathepsin D with a Biotin-labelled pepstatin
| Autor: | C. Graham Knight, Robert S. Decker, Ian T.W. Matthews |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
Biophysics
Biotin Cathepsin D Biochemistry Cathepsin A chemistry.chemical_compound Cathepsin O Structural Biology Pepstatins Genetics medicine Humans Molecular Biology Fluorescent Dyes biology Histocytochemistry Rhodamines Cartilage Synovial Membrane Proteolytic enzymes Cell Biology Fibroblasts Avidin Cathepsins Molecular biology medicine.anatomical_structure Microscopy Fluorescence chemistry biology.protein Oligopeptides Thiocyanates Pepstatin |
| Zdroj: | FEBS Letters. (2):253-256 |
| ISSN: | 0014-5793 |
| DOI: | 10.1016/0014-5793(81)80613-8 |
| Popis: | Proteolytic enzymes are believed to be actively involved in the destruction of articular cartilage which accompanies chronic joint inflammation [ 11. The aspartate proteinase, cathepsin D, is secreted by cultured cartilage stimulated to resorb [2] and there has been considerable interest in the possible role of this enzyme in the degradation of proteoglycans in arthritis [3]. Immunohistochemical methods have demonstrated extracellular cathepsin D at the pannus-articular cartilage junction in rheumatoid arthritic tissue [4]. However, such a procedure does not distinguish between the active and inactive forms of the enzyme. Modified forms of the tight-binding, active-sitedirected inhibitor pepstatin [isovaleryl-L-valyl-Lvalyl-(3S,4S)lt-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-(3S,4S)-4-amino-3-hydroxy-6methylheptanoic acid] could prove useful in localizing cathepsin D [5,6]. Since this class of inhibitor requires the native, active enzyme structure for efficient interaction [7], the binding of this inhibitor can locate potentially active enzyme intraor extracellularly [6]. This report illustrates that a biotinyl-pepstatin, in conjunction with tetramethylrhodaminelabelled avidin, is a useful probe to demonstrate cathepsin D within human synovial fibroblasts. given by Dr G. Evin, INSERM Unite 36, Paris. Biotinyl-N-hydroxysuccinimide was prepared as in [9]. 1,2-Diaminoethane was from Aldrich. Avidin, collagenase type V and bovine trypsin type I were from Sigma. Cathepsin D from human liver was kindly given by Dr A. J. Barrett of this laboratory. Tetramethylrhodamine isothiocyanate was from Polysciences. Sephadex LH-20 and G-25 were from Pharmacia. AG-SOW-X2 resin (H’ form; 200400 mesh) was from BioRad Labs. Plastic Petri dishes and flasks were from Sterilin. Dulbecco’s modified Eagle’s medium and foetal calf serum were from GIBCO. Human synovial cells were prepared from sterile biopsy tissue obtained from the Department of Orthopaedic Surgery, New Addenbrooke’s Hospital, Cambridge. |
| Databáze: | OpenAIRE |
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