Enzymes IC

Welcome Proliferation Cytokines IC Enzymes IC Signal transduction Symposia




Just like cytokines it is easy to proof the presence of enzymes in a cell. By fixation the protein is kept in the cell when this is permeabilized. By using the right antibody the protein can be detected. It is more difficult to show an active enzyme in a cell, because this doesn't allow the fixation.

Surface enzyme activity:
The Activity of dipeptidyl peptidase IV (DPPIV).

It is possible to show enzyme activity on the surface of the cell. In a flow cytometer the activity of the enzyme molecule can be evaluated on different cell populations. Making use of a substrate that has no (detectable) fluorescence and that is converted into a molecule that sticks to lipids (the cell membrane) and has its own characteristic fluorescence the enzyme activity can be detected.
The protocol to detect DPPIV (CD26) is based on a new type of fluorogenic substrate where proteases cut a substrate where one of the leaving groups is cresyl violet. This cresyl violet is not fluorescent when it is attached to amino acids or peptide group(s), but the group becomes highly fluorescent after proteolytic cleavage from this attached group. Its fluorescence shows linearity with concentration and barely any fading. 

Ala-Pro-cresyl violet has properties that it can function as substrate for dipeptidyl peptidase IV (DPPIV) (CD26). The substrate makes it possible to localize and quantify of the  activity of the enzyme in individual  cells using confocal microscopy, image analysis, and flow cytometry. because of it properties the fluorescence of the substrate is localized to the DPPIV (in patches) at plasma membranes.
Cresyl violet-dependent fluorescence can be analyzed by flow cytometry and in a similar way by confocal microscopy. Production of fluorescence can be analyzed on the basis of a series of digital images. By calculation of the initial velocity at time zero, the activity of DPPIV per individual cell can be calculated. 

Boonacker et al., J. Histochem Cytochem, Vol 50(9), 1169 - 1177 (2002)

Although we did our best we can not be held responsible for any mistakes or typing errors.
 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005