Linked Life Data

15230872

Source:http://linkedlifedata.com/resource/pubmed/id/15230872

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
Pt 1
pubmed:dateCreated
2004-7-2
pubmed:abstractText
We describe a technique for imaging enzyme activity through steady-state fluorescence anisotropy measurements on a per-pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 microm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy-FL-labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five-fold. Fluorescence lifetime and time-resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy-FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy-containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0022-2720
pubmed:author
pubmed:issnType
Print
pubmed:volume
215
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
24-33
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy.
pubmed:affiliation
The Institute of Optics, University of Rochester, NY 14642, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.