Tosyl-L-lysine chloromethyl ketone

This ceramide-mediated apoptosis was shown to be inhibited by the simultaneous addition of PKC activators (Ni et at, 1994 Obeid et al, 1993), implying that PS may activate the ceramide-mediated apoptotic pathway. However, the inhibitors of interleukin-1 converting enzyme (ICE)-like proteases (Caspase), such as tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl-L-phenylalanine chloromethyl ketone (TPCK) which inhibit ceramide-mediated apoptosis, had no effect on PS-induced apoptosis (Figure 4). Thus, PS-induced apoptotic pathway appears to be distinct from that mediated by ceramide. Further studies are required to clarify the molecular mechanisms underlying the PS-induced apoptosis. 

TV Tosyl-L-lysine chloromethyl ketone (35-l-chloro-3-tosylamino-7-amino-2-heptanone HCI) [4272-74-6] M 369.3, m 150-153°(dec), 156-158°(dec), -165°(dec), [a]J, -7.3° (c 2, H2O). The hydrochloride slowly crystallises from a cone soln in absolute EtOH, thinned with EtOH-Et2O for collection and dried in vacuo. It is a suicide enzyme inhibitor [Matsuda et al. Chem Pharm Bull Japan 30 2512 1982 Shaw et al. Biochemistry 4 2219 7965]. 

Note Reactions were performed under the conditions described in the text with either colorimetric peptide pNA4 (250 pM) or fluorogenic peptide F3 (35 pM) as a substrate. The IC50 values represent the inhibitor concentration required to reduce the protease activity by 50% of the control containing no inhibitor. NI, no inhibition was observed at the concentrations indicated. E64, frans-epoxysuccinyl-L-leucylamide-(4-guanidino)-butane PMSF, phenylmethylsulphonyl fluoride TLCK, tosyl-L-lysine-chloromethyl ketone. 

TPCK, N-tosylphenylalanine chloromethyl ketone TLCK, N-a-p-tosyl-L-lysine-chloromethyl ketone. 

Tosyl-l-lysine chloromethyl ketone (TLCK) treated a-chymo-trypsine. 

Lysis is carried out in a disrupting device such as a French pressure cell. The preferred lysis buffer contains 100 mM Tris-HCl, pH 8.0 at 25°C, 0.5 M NaCl, 10 toM 2-mercaptoethanol, 50 xg/ml of phenylmethylsulfonyl fluoride (PMSF), 50 p,g/ml of iV-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 50 (ig/ml of IV-a-tosyl-L-lysine chloromethyl ketone (TLCK), 50 (Jig/ml of pepstatin A, 50 p.g/ml of leupeptin, and 1 mM benzamidine. Typically, the cell sample is thawed in a water bath at room temperature in 1 ml of lysis buffer per 2 g of cells, and passed through a French pressure cell at 16,000 psi to produce a lysate. 

Abbreviations used TACK, A "-tosyl-L-arginine chlorometl)yl ketone (L-l-ehloro-3-tosylamido-6-guanidinohexan-2-one) p-NOu-ZACK, W -p-nitrobenayloxycar-bonyl-L-arginine chloromethyl ketone TLCK, W"-tosyl-L-lysine chloromethyl ketone Tos- tosyl Z-, benzyloxycarbonyl- DTTT, dithiothreitol TLME, W -tosyl-L-lysine methyl ester TAME, W -tosyl-L-arginine methyl ester. 

Serine proteases are recognized by their irreversible inhibition by 3,4-dichloroisocoumarin (3,4-DCI), L-3-carboxytrans 2,3-epoxypropyl-leucylamido (4-guanidine) butane (E.64), diisopropylfluorophosphate (DFP), phenylmethanelsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK). Some of the serine proteases are inhibited by thiol reagents such as p-chloromercuribenzoate (PCMB) due to the presence of a cysteine residue near the active site. Serine proteases are generally active at neutral... 

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