Characterization of a serine protease activity in Sarcocystis neurona merozoites.
Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.
Barr
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SC
Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA. scb6@cornell.edu
Warner
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D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004679:Encephalomyelitis / Q000469:parasitology / Q000662:veterinary*; D006734:Horse Diseases / Q000469:parasitology*; D006736:Horses; D008970:Molecular Weight; D012522:Sarcocystis / Q000201:enzymology* / Q000254:growth & development; D012523:Sarcocystosis / Q000469:parasitology / Q000662:veterinary*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013116:Spinal Cord / Q000469:parasitology; D013450:Sulfones / Q000494:pharmacology
D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D012697:Serine Endopeptidases