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position sn-1 and short polar acyl residues in position sn-2 of the glycerol backbone. The respective compounds trigger an intracellular signaling network including sphingomyelinases, (MAP) kinases and transcription factors thereby inducing proliferation or apoptosis of vascular cells. Both phenomena largely depend on the specific action of sphingolipid mediators that are acutely formed upon cell stimulation by the oxidized lipids. Fluorescence microscopic studies on labeled lipid analogs revealed that the short-chain phosphatidylcholines are easily transferred from the aqueous phase into the cell plasma membrane and eventually spread throughout the cells. Under these circumstances, the biologically active compounds are very likely to interfere directly with various signaling components inside the cells, which finally decide about cell growth or death. Investigations are being performed on the levels of the lipidome, the apparent enzyme activities, the proteome an the transcriptome to find the primary molecular targets and their downstream elements that are the key compnents of lipid-induced cell death. 1.2. Oxidative stress and antioxidants Antioxidants protect biomolecules against oxidative stress and thus, may prevent its pathological consequences. Specific fluorescent markers have been established for high-throughput screening of lipid and protein oxidation and its inhibition by natural and synthetic antioxidants. These methods can also be used for the determination of antioxidant capacities of biological fluids (e.g. serum) and edible oils. A prominent example is pumpkin seed oil which contains a variety of antioxidants (e.g. tocopherols and phenolic substances) and other secondary plant components that possess useful biological properties. 2. Functional proteomic analysis of lipolytic enzymes The functional properties of lipases and phospholipases are the subject of our studies in the framework of the joint research program GOLD-Genomics of Lipid-associated Disorders at KFU Graz. Lipolytic enzymes catalyze intra- and extracellular lipid degradation. Dysfunctions in lipid metabolism may lead to various diseases including obesity, diabetes or atherosclerosis. In chemistry, lipases and esterases are important biocatalysts for (stereo)selective reactions on synthetic and natural substrates leading to defined products for pharmaceutical or agrochemical use. 28
2.1. Fluorescent suicide inhibitors for in-gel analysis of lipases and phospholipases Fluorescent suicide inhibitors have been developed as activity recognition probes (ARPs) for qualitative and quantitative analysis of active lipolytic enzymes in complex biological samples (industrial enzyme preparations, serum, cells, tissues). Since inhibitor binding to the active sites of lipases and esterases is specific and stoichiometric, accurate information can be obtained about the type of enzyme and the moles of active protein (active sites) in electrophoretically pure or heterogeneous enzyme preparations. Labelling of enzymes with an ARP specific for serine hydrolases BG Inactive Inactive RG NBD RG: Reacti v e p h osphonate gr o up, irreversibly inhibits the catalytic serine Active BG BG: Binding group: is specifically recognized by the active enzyme Inactive Inactive Inhibited RG NBD NBD : fluorescent tag λ : 488 nm; λ : 540 nm ex em Fluorescent inhibitors are currently applied to proteomic analysis of the lipolytic enzymes in human and animal cells. These studies are performed in the framework of the joint project GOLD (Genomics of Lipid-associated Disorders/ coordinated by KFU Graz) which aims at discovering novel genes, processes and pathways that regulate lipid homeostasis in humans, mice and yeast. This is one of the projects in the field of functional genomics (GEN-AU, GENome research in AUstria) funded by the Austrian Federal Ministry for Education, Science and Culture (bm:bwk). Green: wt Red: ko Red: wt Green: ko DABGE Analysis of lipases and esterases in mouse adipose tissue of wt and lipase ko mice Differential activity-based gel electrophoresis (DABGE) was developed for comparative analysis of two lipolytic proteomes in one polyacrylamide gel. For this purpose, the active 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 29
Page 1 and 2: Graz University of Technology Austr
Page 3 and 4: Brief History of the Institute of B
Page 5 and 6: Highlights of 2010 In 2010, Peter M
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Page 11 and 12: unusual amino acids, i.e. hydroxypy
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Page 15 and 16: 2) Jajcanin-Jozic, N., Deller, S.,
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Graz University of Technology Austria Institute of Biochemistry ...

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