Linked Life Data

12225789

Source:http://linkedlifedata.com/resource/pubmed/id/12225789

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
9
pubmed:dateCreated
2002-9-12
pubmed:abstractText
Activation of caspases is the key event of apoptosis. To detect this event in situ we applied fluorochrome-labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA are fluorescein- or sulforhodamine-conjugated peptide-fluoromethyl ketones that covalently, with 1:1 stoichiometry, bind to enzymatic centers of caspases; the specificity is provided by the peptide sequence of amino acids. Similarly, we applied fluorescent inhibitors of serine proteases (FLISP) to detect active sites of the latter enzymes. Exposure of live cells to FLICA of FLISP led to uptake of these ligands and their binding to activated caspases or active sites of serine proteases; the unbound reagents were removed by cell rinse. Only cells undergoing apoptosis were labeled with FLISP or FLICA. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA or FLISP allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases; two proteins reacting with FLISP were about 57 and 60 kDa, which suggests that they are novel enzymes. Detection of caspases or serine proteases activation can be combined with other markers of apoptosis or cell cycle for multiparametric analysis by flow or laser scanning cytometry. Being caspase inhibitors, FLICA arrest the process of apoptosis and prevent cell disintegration. The stathmo-apoptotic assay was developed, therefore, to obtain cumulative apoptotic index over a long period of time and estimate a rate of cell entry into apoptosis for cell populations.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0301-472X
pubmed:author
pubmed:issnType
Print
pubmed:volume
30
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
982-9
pubmed:dateRevised
2010-12-3
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors.
pubmed:affiliation
Brander Cancer Research Institute at New York Medical College, Valhalla, NY, USA
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Review, Research Support, Non-U.S. Gov't