Purification and characterization of a secreted protease from the pathogenic marine bacterium Vibrio anguillarum.
Vibrio anguillarum is a pathogenic marine bacterium which causes the disease vibriosis in salmonid fish, which is characterized by a fatal hemorrhagic septicemia accompanied by massive tissue destruction. In this paper, the purification of the major caseinolytic extracellular protease from V. anguillarum is presented. The purification steps include ammonium sulfate precipitation, DEAE-Sepharose chromatography, Sephacryl S-200 chromatography, and DEAE high-pressure liquid chromatography. The purified protease migrates with Mr = 38,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A slightly larger protease of Mr 40,000 is also separated by this procedure, but accounts for only a minor fraction of the caseinolytic activity. The Mr 38,000 protease displays a broad pH activity profile in the neutral to basic range. It is not inhibited by serine, cysteine, or acid protease inhibitors, but is inhibited by EDTA and 1,10-phenanthroline, suggesting that it is a metalloprotease. The activity of the EDTA-inactivated protease could be partially restored by the addition of Ca2+ and Zn2+ together. The molecular weight and inhibition data show some similarities with proteases isolated from other Vibrio species such as Vibrio cholerae and Vibrio vulnificus.
Farrell
D H
DH
Department of Microbiology and Immunology, Oregon Health Sciences University, Portland 97201.
Crosa
J H
JH
D000645:Ammonium Sulfate; D002122:Calcium Chloride / Q000494:pharmacology; D011232:Chemical Precipitation; D002712:Chlorides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000302:isolation & purification; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D014733:Vibrio / Q000187:drug effects / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D002712:Chlorides; D011480:Protease Inhibitors; D017967:Zinc Compounds; C016837:zinc chloride; D010450:Endopeptidases; D008666:Metalloendopeptidases; D015032:Zinc; D002122:Calcium Chloride; D000645:Ammonium Sulfate