title: Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites.
abstract: Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.
authors.affiliation: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brasil.
authors.forename: S C
authors.initials: SC
authors.lastname: Alfieri
authors.forename: C E
authors.initials: CE
authors.lastname: Correia
authors.forename: S A
authors.initials: SA
authors.lastname: Motegi
authors.forename: E M
authors.initials: EM
authors.lastname: Pral
mesh_terms: D000048:Acanthamoeba / Q000201:enzymology*; D000818:Animals; D002364:Caseins / Q000378:metabolism; D017077:Culture Media, Conditioned; D003546:Cysteine Endopeptidases / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000032:analysis
chemical_list: D002364:Caseins; D017077:Culture Media, Conditioned; D011480:Protease Inhibitors; C026018:azocasein; D010450:Endopeptidases; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases