Linked Life Data

15113701

Source:http://linkedlifedata.com/resource/pubmed/id/15113701

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2004-4-28
pubmed:abstractText
Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan. It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P. aeruginosa infections. The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S. aureus cell suspensions. This assay, however, does not permit kinetic studies and its reproducibility is poor. Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase. Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader. Activity of nanogram amounts of LasA protease could be determined within a few minutes. Furthermore, this assay permitted the determination of Km and kcat values for LasA protease, which were 0.46 mM and 11.8s(-1), respectively. Pseudomonas elastase was also active in the assay. However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon. The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/4-nitroaniline, http://linkedlifedata.com/resource/pubmed/chemical/Aniline Compounds, http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Endotoxins, http://linkedlifedata.com/resource/pubmed/chemical/Glycopeptides, http://linkedlifedata.com/resource/pubmed/chemical/Metalloendopeptidases, http://linkedlifedata.com/resource/pubmed/chemical/Pancreatic Elastase, http://linkedlifedata.com/resource/pubmed/chemical/Peptides, http://linkedlifedata.com/resource/pubmed/chemical/Protease Inhibitors, http://linkedlifedata.com/resource/pubmed/chemical/phosphoramidon, http://linkedlifedata.com/resource/pubmed/chemical/staphylolysin, http://linkedlifedata.com/resource/pubmed/chemical/staphylolytic protease
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
328
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
225-32
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction.
pubmed:affiliation
Maurice and Gabriela Goldschleger Eye Research Institute, Tel Aviv University Sackler Faculty of Medicine, Sheba Medical Center, Tel-Hashomer 52621, Israel. ekessler@post.tau.ac.il
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.