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.
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 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.
Sawada
K
K
Department of Pharmacology, Okayama University Medical School, Japan.
Nishibori
M
M
Nakaya
N
N
Wang
Z
Z
Saeki
K
K
D000818:Animals; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry / Q000502:physiology; D001930:Brain Injuries / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D007797:Laminin / Q000378:metabolism*; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism*
D005456:Fluorescent Dyes; D007797:Laminin; D011480:Protease Inhibitors; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D003094:Collagen; D012697:Serine Endopeptidases; D014357:Trypsin; D002118:Calcium