Purification and characterization of a trypsin-like serine proteinase fromrat brain slices that degrades laminin and type IV collagen and stimulatesprotease-activated receptor-2

A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammo nium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatograph y, and carboxymethyl-cellulose and gel filtration chromatographies. The gel atinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-p olyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation g ave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphat e, benzamidine, p-toluenesulfonyl-L-lysine chloromethyl ketone, and aprotin in completely inhibited the activity of P22, whereas phenanthroline, p-tolu ene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P2 2 efficiently digested the extracellular matrix proteins laminin and type I V collagen, P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with p rotease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 ca n stimulate protease-activated receptor-2. Rat brain penetration injury ind uced gelatinolytic activity in the lesioned area whose molecular size was c onsistent with that of P22. These results indicated that on incubation of r at brain slices, a trypsin-like serine proteinase was secreted into the med ium that was capable of digesting extracellular matrix and stimulating prot ease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.