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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to Investigate the role of LasB in virulence and LasA processing, a LasB-negative mutant, PA01E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An Internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201–1. The resulting plasmid, pBRMOB-LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB-positive P. aeruginosa strain, PA01. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PA01E supernatant fluids yielded no detectable LasB (〈1 ng ml-1 LasB). The absence of LasB in PA01E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunogiobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PA01E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PA01 and PA01E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastotysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave β-casein. Preliminary analysis of β-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: With the determination of the three-dimensional structure of elastase and the probable identification of the active site and key residues involved in proteolytic activity, our knowledge of the molecular details of this interesting protease is rapidly increasing. Pseudomonas elastase appears to be remarkably similar to the Bacillus metalloproteinase thermolysin. A further significant development has been the discovery of the lasA gene and the fact that Pseudomonas elastase and alkaline proteinase appear to act in concert with the LasA protein to display the notable elastolytic activity exhibited by isolates of this organism. Biochemical and genetic studies indicate that LasA is a second elastase which may be an important virulence factor that has been overlooked in previous studies.
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow-up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat-killed staphylococoi. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N-terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross-reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range, in addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave β-casein.
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