Inhibition of platelet thromboxane synthetase by L-1-tosylamido-2-phenylethyl chloromethyl ketone

L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was found to inhibit several aspects of arachidonic acid (20:4) metabolism in human platelets; the primary effect being inhibition of thromboxane synthetase. Thromboxane B₂ (TxB₂) formation from exogenous 20:4 or PGH₂, or from endogenous 20:4, was inhibited by TPCK at concentrations between 0.1 and 0.5 mM. Formation of malondialdehyde (MDA) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), products which also arise from PGH₂, was inhibited to a similar extent. Inhibition of formation from 20:4 of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), the product of the lipoxygenase pathway, was observed; although the extent of this inhibition was less than that of TxB₂ formation. A small inhibitory effect of TPCK on the release of 20:4 from platelet phospholipids was also observed. This evidence indicated that while a number of reactions are inhibited by TPCK, the primary effect appears to be inhibition of thromboxane synthetase.     

Inhibition of initiation of protein synthesis in mammalian tissue culture cells by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

Incorporation of amino acids into proteins in HeLa cells, virus-transformed 3T3 mouse fibroblasts, and mouse plasmacytoma cells is inhibited after the addition of L-1-tosylamido-2-phenylethyl chloromethyl ketone, an alkylating agent and chymotrypsin-specific protease inhibitor. Addition of this drug to tissue culture cells at concentrations of 20 to 30 mug per ml results in an irreversible inhibition of the incorporation of amino acids into cellular proteins, and a rapid and complete breakdown of polyribosomes. A comparative study examining the effects of L-1-tosylamido-2-phenylethyl chloromethyl ketone and several known inhibitors of in vivo protein synthesis, with known mechanisms of action, revealed that an optimal concentration of L-1-tosylamido-2-phenylethyl chloromethyl ketone: (a) immediately and selectively inhibits initiation of protein synthesis, (b) does not significantly affect normal elongation rates, and (c) does not promote a premature release of nascent peptides. L-1-Tosylamido-2-phenylethyl chloromenthyl ketone may prove to be a useful tool in investigating the initiatior of protein synthesis in eukaryotic cells.     

Lack of inhibition by l-1-chloro-4-phenyl-3-toluene-p-sulphoamidobutan-2-one (l-1-tosylamido-2-phenylethyl chloromethyl ketone) of the formation of bacterial polypeptide chain initiation complex (Short Communication)

The chymotrypsin inhibitor l-1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one does not inhibit the function of the initiation factor during the formation of the polypeptide chain initiation complex in vitro. Since the inhibitor has been shown previously to inhibit polypeptide chain elongation by reacting with elongation factor EF-Tu, the inhibitor can be used to investigate the initiation and elongation steps of protein biosynthesis separately.     

Inhibition of the transformation-specific kinase in ASV-transformed cells by N-alpha-tosyl-L-lysyl chloromethyl ketone.

We find that the protease inhibitor N-α-tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits the transformation-specific kinase activity (Collett and Erikson, 1978) associated with p60^src, the avian sarcoma virus (ASV) gene product responsible for the transformation of fibroblasts. TLCK has been shown to induce the phenotypic reversion of ASV-transformed cells to normal (Weber, 1975). Kinase activity was measured in extracts of chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of ASV (SR-ASV) with antiserum from rabbits bearing ASV-induced tumors. The immunoprecipitates were incubated with γ-³²P-ATP under conditions in which the phosphorylation of the IgG heavy chain in the immunoprecipitate was directly proportional to the concentration of cell extract. When ASV-transformed CEF were treated with 0.1 mM TLCK, the kinase activity was reduced by 60% after 2 hr and by 80% after 6 hr, and continued to remain low for up to 40 hr when TLCK was present. When TLCK was removed, the kinase activity rose slowly over a period of many hours, suggesting that the enzyme is irreversibly inactivated by TLCK and new enzyme must be synthesized. The effect of TLCK in vivo is concentration-dependent and specific. Other serine protease inhibitors had no effect on kinase activity. At low concentrations (0.03 mM), TPCK produced partial inhibition (≤20%), but at higher concentrations TPCK was extremely toxic to the cells and therefore could not be tested. The inhibition by TLCK was not due to its ability to inhibit protein synthesis since cycloheximide treatment (1 μg/ml) did not significantly reduce kinase activity. TLCK also inhibited kinase activity when added directly to cell extracts, but about 5 times higher concentrations of TLCK were required to produce 50% inhibition. Under these conditions both TLCK and TPCK were comparable inhibitors, whereas PMSF had no effect. Our finding that the inhibition of the kinase by TLCK in vivo parallels the reversion of cell morphology to normal suggests that the kinase has an important role in transformation and offers a biochemical rationale for treatment of tumors with this agent.     

Differential inhibition of globin chain synthesis by tosyl-L-lysyl chloromethyl ketone

Tosyl-L-lysyl chloromethyl ketone (TLCK), added to an incubation of rabbit reticulocytes, inhibits the synthesis of α globin chain more than that of β chain. TLCK has been previously shown to inhibit initiation of translation in a variety of cells. Thus this drug could be used to test for such a differential effect at the level of peptide initiation on the various mRNAs in these cells.     

Inhibition of macromolecular synthesis by caffeine in Clostridium perfringens

Caffeine (2 mg/mL) inhibited the incorporation of [14C]adenine into actively growing cells of Clostridium perfringens NCTC 8679 in a dose-dependent manner. Also reduced by caffeine was incorporation of [14C]thymidine and 14C-labeled amino acids. No effect on guanine, uracil, adenosine, guanosine, or uridine was detected. Actual incorporation of [14C]caffeine or [14C]thymine in control cultures did not occur.     

Regulation of macromolecular synthesis in reovirus-infected L-929 cells I. Effect of L-histidinol.

The histidine analogue L-histidinol, reported by Vaughan and Hansen (1973) to establish a potent, readily reversible inhibition of eukaryotic protein synthesis in vivo, was used to investigate the regulation of macromolecular synthesis in reovirus-infected L-929 cells. The addition of L-histidinol to normal L cells led to a total inhibition of protein synthesis. The inhibition appeared to be a consequence neither of isotope dilution resulting from elevated endogenous amino acids nor of an inability of treated cells to accumulate exogenous amino acids. Addition of L-histidine to histidinol-arrested cells resulted in a complete recovery of protein synthesis. Similarly, protein synthesis in reovirus-infected L cells examined 17 h postinfection (31 C) was totally inhibited by histidinol treatment and was readily reversed by the addition of histidine. Reovirus-infected cells treated with histidinol had an essentially unaltered capacity to synthesize reovirus single-stranded RNA relative to unperturbed cultures but a diminishing ability to maintain genome RNA synthesis. Addition of L-histidine to arrested cultures led to a complete recovery of genome RNA synthesis. The L-histidinol-mediated arrest of protein synthesis was both very effective and easily reversed, suggesting the general applicability of this novel inhibitor to investigations of regulation of macromolecular synthesis in both normal and virus-infected eukaryotic cells.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

Long-lasting antinociceptive effect of DAMGO chloromethyl ketone in rats

Previously, the opioid peptide Tyr-D-Ala-Gly-(NMe)Phe-CH2Cl (DAMCK) has been shown to bind irreversibly to mu opioid receptors in vitro. In the present work, the antinociceptive effect of DAMCK has been evaluated. Rats treated systemically with DAMCK (1-100 pg/kg) displayed a dose-dependent increase in tail-flick analgesia that peaked by 15 min, then stayed about the same until 60 min, followed by some decrease over time. Higher doses of DAMCK (10 ng/kg-100 microg/kg) produced a near-maximal antinociceptive effect that remained stable for 4 h. Significant antinociception was still detected 8 h, but not 24 h postinjection. In comparison, the parent peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) reached maximal effect by about 30 min, followed by a rapid cessation of its antinociceptive response. Naloxone administered before DAMCK antagonized the antinociceptive response of DAMCK, indicating that it was mediated via opioid receptors. Naloxone administered 45 min after DAMCK attenuated the tail-flick response to some extent, but a substantial part (40-60% depending on the peptide concentration and evaluation time) remained unaffected. Central administration of DAMCK also elicited time- and concentration-dependent, profound, opioid receptor mediated, apparently irreversible antinociception.     

Inactivation of the plasminogen activator from HeLa cells by peptides of arginine chloromethyl ketone.

The binding specificities of human urinary urokinase (EC 3.4.99.26) and HeLa cell plasminogen activator were studied using peptidyl chloromethyl ketone inhibitors. A 125I-labeled fibrin assay has been developed to yield kinetic information. Reagents of the sequence X-Gly-ArgCH2Cl were the most effective. The susceptibility of the HeLa cell plasminogen activator differed from that of urokinase in several respects indicating the utility of this type of inhibitor in distinguishing between proteases of this specificity.