tosylphenylalanyl-chloromethyl-ketone and 1-10-phenanthroline

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.

Topics: Aedes; Animals; Larva; Molecular Weight; Pepstatins; Phenanthrolines; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Pupa; Serine Endopeptidases; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.

Topics: Analysis of Variance; Animals; Gametogenesis; Leupeptins; Mice; Mice, Inbred BALB C; Phenanthrolines; Phenylmethylsulfonyl Fluoride; Plasmodium berghei; Protease Inhibitors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Zygote

Proteinase inhibitors were evaluated for their anti-inflammatory actions on carrageenin-induced inflammation in rats. The development of granulation tissue and the exudate were markedly suppressed by a single injection of L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) into the carrageenin-air-pouch immediately after carrageenin injection, whereas a single injection of TPCK at 12 or 24 hr after carrageenin injection was less effective or slightly effective respectively. These results suggest that proteinase inhibitors exert their anti-inflammatory actions by interfering with the initial inflammatory reactions after carrageenin injection. When the wet weight of granulation tissue and the weight of exudate were measured on day 4 after the simultaneous injection of carrageenin and inhibitors, a single injection of serine- and thiol-proteinase inhibitors including TPCK, leupeptin, antipain, chymostatin and cystamine suppressed the development of granulation tissue, though EDTA and o-phenanthroline, metallo-proteinase inhibitors, were also effective at a high dose. Exudate was reduced by treatment with TPCK in a dose-dependent manner, while EDTA and o-phenanthroline were effective only at a high dose. On the other hand, the migration of polymorphonuclear leukocytes into the carrageenin-air-pouch (the inflammatory lesion) was markedly suppressed by TPCK and leupeptin, while a high dose of cystamine and o-phenanthroline was slightly effective, and antipain, chymostatin, pepstatin, elastatinal, EDTA, trans-1-aminomethylcyclohexane 4-carboxylic acid and aprotinin were without effect.

Topics: Animals; Carrageenan; Cell Movement; Cystamine; Inflammation; Leupeptins; Male; Neutrophils; Phenanthrolines; Protease Inhibitors; Rats; Rats, Inbred Strains; Tosylphenylalanyl Chloromethyl Ketone