Measurement of protease activity of live Uronema marinun (Ciliata: Scuticociliatida) by fluorescence polarization.
The proteolytic activity of live Uronema marinum was analyzed by a fluorescence polarization (FP) technique. Protease activity was measured as a decrease in the FP value using fluorescein isothiocynate (FITC)-casein as a protein substrate. A time-dependent decrease in FP occurred in plate wells containing live U. marinum. Supplementation with the cysteine protease inhibitor E-64 had no significant inhibitory effect on the decrease in FP at any of the concentrations used. In contrast, supplementation with 1,10-phenanthroline resulted in complete inhibition of proteolysis for 30 min at 1 mM and for 1 h at 2 and 5 mM. Effective inhibition of the proteolytic activity of live U. marinum by 1,10-phenanthroline indicated that metalloproteases are the main proteases excreted by U. marinum. As U. marinum has a high potential for systemically invading and destroying fish tissues, the metalloproteases excreted by live U. marinum are likely to be involved in the invasion of host tissues and the pathogenicity of the parasite.
Lee
Eun Hye
EH
Department of Aquatic Life Medicine, Pukyong National University, Pusan 808-737, Korea.
Kim
Chun Soo
CS
Cho
Jae Bum
JB
Ahn
Kyoung Jin
KJ
Kim
Ki Hong
KH
D000818:Animals; D016798:Ciliophora / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization / Q000379:methods / Q000662:veterinary*; D005456:Fluorescent Dyes; D008666:Metalloendopeptidases / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*
D005456:Fluorescent Dyes; D010618:Phenanthrolines; D011480:Protease Inhibitors; D010450:Endopeptidases; D008666:Metalloendopeptidases; D016650:Fluorescein-5-isothiocyanate; C025205:1,10-phenanthroline