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- Title
Purification and Characterization of a Trypsin-Like Serine Proteinasefrom Rat Brain Slices that Degrades Laminin and Type IV Collagen andStimulates Protease-Activated Receptor-2.
- Authors
Sawada, Ken; Nishibori, Masahiro; Nakaya, Naoki; Wang, Zhao; Saeki, Kiyomi
- Abstract
Abstract: A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatography, and carboxymethyl-cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphate, benzamidine, p-toluenesulfonyl-L-lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p-toluene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca[sup 2+] concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 can stimulate protease-activated receptor-2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin-like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22...
- Subjects
TRYPSIN; SERINE proteinases; EXTRACELLULAR matrix; BRAIN; CYTOCHEMISTRY
- Publication
Journal of Neurochemistry, 2000, Vol 74, Issue 4, p1731
- ISSN
0022-3042
- Publication type
Article
- DOI
10.1046/j.1471-4159.2000.0741731.x