Purification and characterization of two high-molecular-mass forms of Achromobacter collagenase

Tong, Nguyen Thanh; Tsugita, Akira; Keil-Dlouhá, V.

doi:10.1016/0167-4838(86)90028-2

Cited by 22 publications

(23 citation statements)

References 28 publications

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“…24.8) have been purified and well characterized (Tong et al, 1986). The DNA encoding the collagenase of V .…”

Section: Discussionmentioning

confidence: 99%

Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli

Yu¹,

Lee²

1999

Microbiology

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The prtV gene, encoding a collagenase of Vibrio parahaemolyticus, was expressed in Escherichia coli and purified by affinity chromatography. The transformant E. coli BL2l (DE3)(pPRT2) secreted the recombinant Prtv, and the highest enzyme activity was detected in the culture supernatant after 5 h IPTG induction. The molecular mass o f purified PrtV was 62 kDa as determined by gel filtration, which was similar to that obtained by SDS-PAGE (64 kDa). This suggested that PrtV was a monomer protein having no subunit structure. The isoelectric point o f PrtV was 852. In addition, PrtV contained a 27 amino acid signal peptide, and the amino acid composition of the PrtV showed satisfactory agreement with that predicted from the DNA sequence. The optimum temperature and pH of PrtV were 40 OC and pH 7.5, respectively. The activity of PrtV was inhibited by chelators such as EDTA, EGTA and 1,lOphenanthroline; however, its activity was restored by the addition of various metal ions (Co2+, Mn2+, Ca2+, Cu2+, Ni2+ and Zn2+), indicating that PrtV is a metalloprotease. PrtV degraded both type I collagen and synthetic substrate FALGPA well, showing that PrtV is indeed a collagenase.

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“…24.8) have been purified and well characterized (Tong et al, 1986). The DNA encoding the collagenase of V .…”

Section: Discussionmentioning

confidence: 99%

Expression and characterization of the prtV gene encoding a collagenase from Vibrio parahaemolyticus in Escherichia coli

Yu¹,

Lee²

1999

Microbiology

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“…5). We have not yet determined the exact C-terminal region, since we have not performed deletion mutation experiments (17) and three possible molecular masses, i.e., 70, 90, and 110 kDa, have been proposed (14). To resolve these issues, it is essential to first determine the complete DNA sequence of the collagenase structural gene.…”

Section: And Discussionmentioning

confidence: 99%

“…Both the culture method of Vibrio alginolyticus chemovar iophagus (V. alginolyticus) and the method for purification of collagenase were described previously (6,7,10). Anti-collagenase antibody was produced in rabbits by three 250 ,ug injections per animal of 90 kDa collagenase, which had been further purified using an HPLC column TSK-DEAE35W (14). The anti-collagenase antibody was detected at 16 x dilution by Ouchterlony assay .…”

Section: Methodsmentioning

confidence: 99%

“…This strain was later classified as Vibrio alginolyticus chemovar iophagus (V. alginolyticus) (1). The structure and functional properties of this Vibrio collagenase (EC 3.4.24.8) have been described in a series of papers by B. Keil and his colleagues (4,6,9,11,13,14). In addition, this enzyme is of practical importance because it is very effective in promoting healing of abnormal skin scars, and can tenderize meat by cleaving collagen.…”

mentioning

confidence: 99%

“…Three active forms of the collagenase have been isolated from the culture supernatant of the organism, which have molecular masses of 110, 90, and 70 kilodaltons (kDa), respectively. These active forms are assumed to be cleaved products derived from the same simple polypeptide chain (14). The molecular weight of the native collagenase remains unknown.…”

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confidence: 99%

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