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