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-toluene-sulfonyl-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 Ca2+ 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.
| Original language | English |
|---|---|
| Pages (from-to) | 1731-1738 |
| Number of pages | 8 |
| Journal | Journal of Neurochemistry |
| Volume | 74 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2000 |
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Keywords
- Brain injury
- Extracellular matrix
- Protease-activated receptor-2
- Serine proteinase
- Trypsin
ASJC Scopus subject areas
- Biochemistry
- Cellular and Molecular Neuroscience
Cite this
Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2. / Sawada, Ken; Nishibori, Masahiro; Nakaya, Naoki; Wang, Zhao; Saeki, Kiyomi.
In: Journal of Neurochemistry, Vol. 74, No. 4, 2000, p. 1731-1738.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2
AU - Sawada, Ken
AU - Nishibori, Masahiro
AU - Nakaya, Naoki
AU - Wang, Zhao
AU - Saeki, Kiyomi
PY - 2000
Y1 - 2000
N2 - 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-toluene-sulfonyl-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 Ca2+ 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.
AB - 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-toluene-sulfonyl-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 Ca2+ 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.
KW - Brain injury
KW - Extracellular matrix
KW - Protease-activated receptor-2
KW - Serine proteinase
KW - Trypsin
UR - http://www.scopus.com/inward/record.url?scp=0034022706&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034022706&partnerID=8YFLogxK
U2 - 10.1046/j.1471-4159.2000.0741731.x
DO - 10.1046/j.1471-4159.2000.0741731.x
M3 - Article
C2 - 10737632
AN - SCOPUS:0034022706
VL - 74
SP - 1731
EP - 1738
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 4
ER -