Purification and characterization of novel trypsin-like serine proteases from mouse spleen.
Novel trypsin-like serine proteases (mouse trypsin-type serine proteases 1 and 2 [MTSP-1 and -2]) were purified to homogeneity from mouse spleen. Each protease consisted of a single polypeptide with a molecular mass of about 29 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Both were totally inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, aprotinin, antipain, and leupeptin and partially inhibited by chymostatin and dithiothreitol, suggesting that they are trypsin-like serine proteases. They hydrolyzed synthetic substrates for trypsin-like proteases but not those for chymotrypsin-like proteases, elastase and kallikrein. MTSP-1 hydrolyzed tert-butyloxycarbonyl (Boc)-Asp(OBzl)Pro-Arg-amino-4-methyl-coumaryl-7-amide (MCA) and Boc-Ile-Glu-Gly-Arg-MCA faster than Boc-Phe-Ser-Arg-MCA. On the other hand, MTSP-2 hydrolyzed Boc-Phe-Ser-Arg-MCA most rapidly, with a specific activity 15 times higher than that of MTSP-1. The N-terminal amino acid sequence of MTSP-1 was Ile-Val-Gly-Gly-Tyr-Thr-His-Leu-Asp-Asn-Gln-Val-Pro-Tyr. This sequence was 71% homologous with the N-terminal of bovine trypsin. The Boc-Phe-Ser-Arg-MCA hydrolyzing activity of mouse spleen significantly (p < 0.01) increased to about 1.5-fold the basal activity 2 weeks after an injection of Freund's complete adjuvant, suggesting that these proteases are involved in the immune response.
Fukusen
N
N
Department of Biochemistry and Nutrition, National Institute of Public Health, Tokyo.
Aoki
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005620:Freund's Adjuvant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013154:Spleen / Q000201:enzymology* / Q000276:immunology; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry
D003374:Coumarins; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; C048055:tertiary-butyloxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumarin-7-amide; D005620:Freund's Adjuvant; C101392:MTSP-1 serine protease; C101393:MTSP-2 serine protease; D012697:Serine Endopeptidases; D014357:Trypsin