Staff Members of the Institute of Biochemistry, TU - Institut für ...

lipases/esterases of two different samples are labelled with fluorescent inhibitors that possess
identical substrate analogous structures but carry different cyanine dyes as reporter
fluorophores. After sample mixing and protein separation by 1-D or 2-D PAGE, the enzymes
carrying the sample-specific colors are detected and quantified. This technique can be used
for the determination of differences in enzyme patterns, e.g. due to effects of genetic
background, environment, metabolic state and disease.
2.2. Protein microarrays – Enzyme chips
In the framework of the Kplus Research Centre
Applied Biocatalysis (TU Graz), we have
developed "biochips" representing ordered
microarrays of biospecific ligands for the detection
and quantification of lipolytic enzymes in
biological samples. In addition, microarrays of
enzymes are generated to determine protein
affinities for substrate analogs. The development
of these tools basically requires three steps, namely
specific loading of activated supports with
bioaffinity ligands, measurement of lipase binding
(e.g. by fluorescence techniques), and application
30
Angewandte
Biokatalyse
Kompetenzzentrum GmbH
of the bio chips to the investigation of real analytical problems. These systems can be used
for the screening of lipases with specific substrate acceptance.
Diploma thesis completed:
Bojana Stojcic: Fluorescence imaging of oxidized phospholipid uptake into cultured
macrophages.
Plasma lipoproteins are oxidized as a consequence of oxidative stress (enzymatic and
nonenzymatic), which in the end can cause lipid and protein modifications of the particles.
Lipid modifications can induce generation of oxidized phospholipids (oxPL) primarily from
(poly)unsaturated diacyl- and alk(en)ylacyl glycerophospholipids including
palmitoyloxovaleroyl-PC (POVPC) and palmitoylglutaroyl-PC (PGPC). Fluorescent analogs
of POVPC and PGPC labeled with BODIPY (POVPE-BY and PGPE-BY, respectively) were
used as tools for studying uptake and intracellular stability of these compounds in cultured
macrophages. The aim of this study was to understand in more detail how oxPL are delivered
to the cells and to analyze their metabolic fate inside the cells. The emphasis was on the role
of the (supra)molecular carriers delivering oxPL to the cells including lipid micelles, albumin
and (mm)LDL. The import of the fluorescent lipids from these donors into macrophages was
studied using fluorescence microscopy. In addition, we analyzed the transfer of oxPL between
albumin and LDL and the degradation products of PGPE-BY as well as the complexes of the
chemically reactive POVPE-BY with proteins or lipids. From our results, it can be inferred
that POVPC mainly elicits its biological effects as a conjugate with lipids and/or proteins. In
order to understand the biological activites of PGPC, we have to take into account that they
are mediated by various compounds including the intact oxPL and its metabolites..