Tosyl-lysine chloromethyl ketone

Figure 1. Processing of picomavirus. polyprotein by proteolytic enzymes. The numbers in brackets represent molecular weights in thousands. Insert Sodium dodecyl sulfate/ polyacrylamide gel electrophoresis of poliovirus polypeptides, labelled with 5% methionine in infected HeLa cells, a. Infected, labelled at 2.5 hours post infection, b. Labelled in presence of 0.5 niM iodoacetamide. c. Labelled in presence of 0.1 mM tosyl 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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