title: Characterization of a serine protease activity in Sarcocystis neurona merozoites.
abstract: 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.
authors.affiliation: Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA. scb6@cornell.edu
authors.forename: S C
authors.initials: SC
authors.lastname: Barr
authors.forename: K
authors.initials: K
authors.lastname: Warner
mesh_terms: 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
chemical_list: D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D012697:Serine Endopeptidases