Purification and characterization of a Clostridium perfringens 120- kilodalton collagenase and nucleotide sequence of the corresponding gene

Osamu Matsushita, K. Yoshihara, S. I. Katayama, J. Minami, A. Okabe

Research output: Contribution to journalArticle

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Abstract

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from ~120 to ~80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.

Original languageEnglish
Pages (from-to)149-156
Number of pages8
JournalJournal of Bacteriology
Volume176
Issue number1
Publication statusPublished - 1994
Externally publishedYes

Fingerprint

Clostridium perfringens
Collagenases
Enzymes
Genes
Colicins
lysyl endopeptidase
Amino Acid Sequence
Vibrio alginolyticus
Amino Acids
Reading Frames
Peptides
Protein Sorting Signals
Collagen Type I
Sodium Dodecyl Sulfate
Vertebrates
Zinc
Polyacrylamide Gel Electrophoresis
Escherichia coli

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Purification and characterization of a Clostridium perfringens 120- kilodalton collagenase and nucleotide sequence of the corresponding gene. / Matsushita, Osamu; Yoshihara, K.; Katayama, S. I.; Minami, J.; Okabe, A.

In: Journal of Bacteriology, Vol. 176, No. 1, 1994, p. 149-156.

Research output: Contribution to journalArticle

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