Functional domains of a zinc metalloprotease from Vibrio vulnificus

Shin-ichi Miyoshi, H. Wakae, K. I. Tomochika, S. Shinoda

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

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 coil DH5α 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°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 K(m) 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.

Original languageEnglish
Pages (from-to)7606-7609
Number of pages4
JournalJournal of Bacteriology
Volume179
Issue number23
Publication statusPublished - 1997

Fingerprint

Vibrio vulnificus
Metalloproteases
Zinc
Peptide Hydrolases
Erythrocyte Membrane
Peptides
Proteins
Escherichia
Oligopeptides
Ammonium Sulfate
Wound Infection
Proteolysis
Chromatography
Sepsis
Membrane Proteins
Plasmids
Erythrocytes
Rabbits
Genes

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Miyoshi, S., Wakae, H., Tomochika, K. I., & Shinoda, S. (1997). Functional domains of a zinc metalloprotease from Vibrio vulnificus. Journal of Bacteriology, 179(23), 7606-7609.

Functional domains of a zinc metalloprotease from Vibrio vulnificus. / Miyoshi, Shin-ichi; Wakae, H.; Tomochika, K. I.; Shinoda, S.

In: Journal of Bacteriology, Vol. 179, No. 23, 1997, p. 7606-7609.

Research output: Contribution to journalArticle

Miyoshi, S, Wakae, H, Tomochika, KI & Shinoda, S 1997, 'Functional domains of a zinc metalloprotease from Vibrio vulnificus', Journal of Bacteriology, vol. 179, no. 23, pp. 7606-7609.
Miyoshi, Shin-ichi ; Wakae, H. ; Tomochika, K. I. ; Shinoda, S. / Functional domains of a zinc metalloprotease from Vibrio vulnificus. In: Journal of Bacteriology. 1997 ; Vol. 179, No. 23. pp. 7606-7609.
@article{d1c96ad6b2034b22b3864cc8c6d23cc9,
title = "Functional domains of a zinc metalloprotease from Vibrio vulnificus",
abstract = "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 coil DH5α 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°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 K(m) 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.",
author = "Shin-ichi Miyoshi and H. Wakae and Tomochika, {K. I.} and S. Shinoda",
year = "1997",
language = "English",
volume = "179",
pages = "7606--7609",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "23",

}

TY - JOUR

T1 - Functional domains of a zinc metalloprotease from Vibrio vulnificus

AU - Miyoshi, Shin-ichi

AU - Wakae, H.

AU - Tomochika, K. I.

AU - Shinoda, S.

PY - 1997

Y1 - 1997

N2 - 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 coil DH5α 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°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 K(m) 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.

AB - 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 coil DH5α 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°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 K(m) 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.

UR - http://www.scopus.com/inward/record.url?scp=0030704251&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030704251&partnerID=8YFLogxK

M3 - Article

C2 - 9393733

AN - SCOPUS:0030704251

VL - 179

SP - 7606

EP - 7609

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 23

ER -