Characterization of purified metallo- and cysteine proteases from the pathogenic haemoflagellate Cryptobia salmositica Katz 1951.
The purified metalloprotease and the partially purified cysteine protease from pathogenic Cryptobia salmositica were characterized. Using haemoglobin gel electrophoresis, we detected five enzymatic bands in crude parasite lysate; one protease (200 kDa) yielded a metalloprotease band and other four, cysteine protease bands (97, 70, 66 and 49 kDa). Both the metalloprotease and the cysteine protease had high levels of proteolytic activity against azocasein, haemoglobin and fibrinogen. The metalloprotease had high levels of activity against azocoll and gelatin but a low degree of activity against albumin. In contrast, the cysteine protease had extensive activity against albumin but low levels of activity against azocoll and gelatin. The metallo- and cysteine proteases had no activity against Pz-peptide, a specific substrate for bacterial collagenase. The optimal pH for the metalloprotease and the cysteine protease was 7.0 and 5.0, respectively. The metalloprotease was inhibited by metal-chelating agents and excess of zinc ions but was activated by calcium ions. The cysteine protease was inhibited by thiol-blocking agents. The natural antiprotease alpha2-macroglobulin, but not alpha1-protease inhibitor, inhibited the activity of both proteases from C. salmositica. The optimal in vitro temperature for the purified metalloprotease was 30 degrees C.
Zuo
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Department of Zoology, University of Guelph, Ontario, Canada.
Woo
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D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D016830:Kinetoplastida / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D010618:Phenanthrolines; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases; D015032:Zinc; D002118:Calcium; C025205:1,10-phenanthroline