Enhancement of catalytic activities of serine proteases by tripeptides compounds

Abstract

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), n-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr- Materials Trp (tPTW), were obtained by screening of synthetic peptides forBovine pancreatic trypsin and chymotrypsin, bovine plasma thromgrowth-inhibitory activity toward cultured transformed cells. The bin, bovine plasma plasmin, porcine pancreastic elastase, human kideffects of these peptide compounds on proteases were investigated ney urokinase, bovine spleen cathepsin B, papain, and porcine kidney and the results showed that these compounds enhanced the leucine aminopeptidase (LAPase)were purchased from Sigma Chemiamidolytic activity of serine proteases despite the fact that each cal Co. (St. Louis, MO). Porcine erythrocyte m-and ~t-calpains were reaction was carried out under optimal conditions. ERPm obtained from Nakarai Chemicals (Kyoto, Japan). The substrates of proteases were obtained from Peptide Institute Inc. (Osaka, Japan). stimulated the activities of trypsin, chymotrypsin, thrombin, Peptide compounds were synthesized by conventional liquid phase plasmin urokinase and elastase, dPTWm also showed similar fragment condensation and purified as described previously [1]. The effects except that toward chymotrypsin, tPTW elevated the purity of each peptide was analyzed by thin-layer or high-performance activity only of trypsin, chymotrypsin and thrombin. Stimulation liquid chromatography. NMR spectroscopy, elemental analysis and of trypsin activity by these compounds was also confirmed by specific rotation. All peptides were dissolved in dimethylsulfoxide using casein as a substrate. None of these compounds affected the (DMSO) at a concentration of 20 mg/ml. amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m-and ~t-calpains, cathepsin B and papain) or an exopeptidase 0eucine aminopeptidase). The Assay forprotease activityAmidolytic activities of serine and cysteine proteases were measured activation was at least partly due to the stabilization of the by incubation at 37°C for 10 min in the following reaction mixture catalytic activity of proteases as well as prevention of autolysis.(0.1 ml); trypsin (100 ng), 1 x PBS, 1 mM EDTA and 10 ~tM Boc-PheSer-Arg-MCA; chymotrypsin (25 ng), 1 XPBS and 10 ~tM Suc-LeuKey words." Serine protease; Protease-stimulatory tripeptide;Leu-Val-Tyr-MCA; thrombin (1.1 ng), 50 mM Tris-HC1 (pH 7.5), 150 Synthetic tripeptide mM NaC1, 2.5 mM CaC12 and 10 laM Boc-Val-Pro-Arg-MCA; plasrain (20 lag), 1 x PBS and 10 laM Boc-Val-Leu-Lys-MCA; elastase (15 /.tg), 50 mM Tris-HC1 (pH 8.8), 1 mM EDTA and 10 laM Suc-AlaPro-Ala-MCA; urokinase (1 munit), 1XPBS, 1 mM EDTA and 10 1. Introduction gM Pyr-Gly-Arg-MCA; cathepsin B (10 launits), 50 mM MES (pH 6.0), 2 mM EDTA, 2 mM DTT and 10 ~tM Z-Arg-Arg-MCA; papain (2.5 ng), 50 mM MES (pH 5.5), 2 mM EDTA, 2 mM DTT and 10 We have previously reported the results of screening of biolaM Z-Phe-Arg-MCA; LAPase (100 launits), 50 mM Tris-HC1 (pH active peptides for growth-inhibitory activity toward trans-7.4), 5 m...

Induction of apoptosis by a calpain stimulator, ONO-3403

“…~4 We have also found that tripeptide compounds such as thioproline-Thr-Trp increase the proteolytic activity of trypsin. 15 It has also been reported that a protein named regucalcin enhanced the proteolytic activity of calpain. ~6 It is thus possible that there are many protease-stimulators of which the action mechanism remains to be proved.…”