Functional domains of a zinc metalloprotease from Vibrio vulnificus.
Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, secretes a 45-kDa metalloprotease (V. vulnificus protease; VVP). A plasmid which carries the entire vvp gene subcloned into pBluescriptIIKS+ was transformed into Escherichia coli DH5alpha for overproduction of the protease. The 45-kDa recombinant protease (rVVP) was isolated from the periplasmic fraction of the transformant by ammonium sulfate precipitation followed by column chromatography on phenyl Sepharose. Biochemical characterization of the isolated rVVP showed that the recombinant protease was identical to that produced by V. vulnificus. When rVVP was incubated at 37 degrees C, a 35-kDa fragment was generated through autoproteolytic removal of the C-terminal peptide. This 35-kDa fragment (rVVP-N) was found to have sufficient proteolytic activity toward oligopeptides and soluble proteins but had markedly reduced activity toward insoluble proteins. Lineweaver-Burk plot analysis indicated increased Km values of rVVP-N for all of the protein substrates. rVVP, but not rVVP-N, was shown to agglutinate rabbit erythrocytes, bind to the erythrocyte ghosts, and digest the ghost membrane proteins. These results strongly suggest that rVVP (and VVP) consists of at least two functional domains: an N-terminal 35-kDa polypeptide mediating proteolysis and a C-terminal 10-kDa polypeptide which may be essential for efficient attachment to protein substrates and erythrocyte membranes.
Miyoshi
S
S
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-Naka, Japan. miyoshi@pheasant.pharm.okayama-u.ac.jp
Wakae
H
H
Tomochika
K
K
Shinoda
S
S
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D006384:Hemagglutination; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology*; D015032:Zinc
D010446:Peptide Fragments; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D015032:Zinc