Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin
✍ Scribed by Krister Karlsson; Fred Nyberg
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 55 KB
- Volume
- 11
- Category
- Article
- ISSN
- 0952-3499
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✦ Synopsis
An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe 7 -Phe 8 residues was purified from the membrane-bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP-hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy-terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X-100 in 20 mM Tris-HCl pH 7.8. After ion exchange chromatography (DEAE-Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G-50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex 1 75 HR 10/30) using a new preparative system, A ¨KTA TM -purifier, controlled by UNICORN 1 software version 2.20.