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Untersuchungen Zum Einfluss Von Proteasen Auf Dieil-6

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Untersuchungen Zum Einfluss Von Proteasen Auf Dieil-6 Untersuchungen von pflanzlichen Latices hinsichtlich ihrer Proteaseaktivität und deren Einfluss auf die Interleukin-6 Sekretion monozytischer Zellen Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Apotheker André Domsalla Berlin 2012 Erster Gutachter: Prof. Dr. Matthias F. Melzig Zweiter Gutachter: Prof. Dr. Harshadrai M. Rawel Disputation am: 25.04.2012 Für meine Familie I Abkürzungsverzeichnis ............................................................................................................ VI I. Einleitung ................................................................................................................................ 1 I.1. Proteasen .......................................................................................................................... 1 I.1.1. Klassifizierung proteolytischer Enzyme .................................................................... 1 I.1.1.1. Serinproteasen .................................................................................................... 2 I.1.1.2. Cysteinproteasen ................................................................................................ 3 I.1.1.3. Aspartatproteasen ............................................................................................... 3 I.1.1.4. Metalloproteasen ................................................................................................ 3 I.1.2. Vorkommen und Bedeutung von Pflanzenmilchsäften ............................................. 3 I.1.2.1. Industriell genutzte Milchsäfte .......................................................................... 5 I.1.2.2. Pharmazeutische und traditionelle Nutzung von Pflanzenmilchsäften .............. 6 I.1.3. Proteolytische Enzyme in Pflanzenmilchsäften......................................................... 6 I.1.3.1. Serinproteasen aus Milchsäften ......................................................................... 7 I.1.3.2. Cysteinproteasen aus Milchsäften ...................................................................... 8 I.1.3.3. Aspartatproteasen aus Milchsäften .................................................................. 10 I.1.3.4. Metalloproteasen aus Milchsäften ................................................................... 10 I.1.3.5. Proteasehaltige Milchsäfte ohne Zuordnung.................................................... 11 I.1.4. Euphorbiaceae.......................................................................................................... 12 I.1.4.1. Systematischer Überblick Euphorbiaceae ........................................................ 12 I.1.4.2. Gattung Euphorbia ........................................................................................... 13 II. Zielsetzung der Arbeit ......................................................................................................... 15 III. Material und Methoden ...................................................................................................... 16 III.1. Geräte ........................................................................................................................... 16 III.1.1. Elektrophorese ...................................................................................................... 16 III.1.2. Zellkultur ............................................................................................................... 16 III.1.3. Waagen.................................................................................................................. 16 III.1.4. Zentrifugen ............................................................................................................ 16 III.1.5. Sonstige Geräte ..................................................................................................... 16 III.2. Verbrauchsmaterial ...................................................................................................... 17 III.2.1. Zellkultur ............................................................................................................... 17 III.2.2. Elektrophorese ...................................................................................................... 18 III.2.3. Substrate ................................................................................................................ 19 III.2.4. Enzyme ................................................................................................................. 19 III.2.5. Inhibitoren ............................................................................................................. 19 III.2.6. Dialyse .................................................................................................................. 20 II III.2.7. Chromatographie ................................................................................................... 20 III.2.8. Zelllinien ............................................................................................................... 20 III.2.9. Kits ........................................................................................................................ 20 III.3. Puffer und Chemikalien ............................................................................................... 21 III.4. Verbrauchmaterialien .................................................................................................. 22 III.5. Programme ................................................................................................................... 23 III.6. Methoden ..................................................................................................................... 24 III.6.1. Probengewinnung und Latexaufbereitung ............................................................ 24 III.6.2. Protein-Bestimmung ............................................................................................. 24 III.6.2.1. Bradford-Assay ............................................................................................. 24 III.6.2.2. Bicinchoninsäure Assay (BCA-Assay) ......................................................... 24 III.6.3. Protease Assays ..................................................................................................... 25 III.6.3.1. Universelles Protease Substrat Roche® ........................................................ 25 III.6.3.2. Enzchek® Protease Assay Kit Green Fluoreszenz ......................................... 26 III.6.4. Untersuchungen zur Lagerungsstabilität ............................................................... 27 III.6.5. Bestimmung der Residualaktivitäten nach Zugabe spezifischer Inhibitoren ........ 28 III.6.6. Proteinkonzentrierung ........................................................................................... 29 III.6.7. Untersuchungen hinsichtlich der Substratspezifität .............................................. 29 III.6.7.1. Synthetische Substrate ................................................................................... 29 III.6.7.2. In-Gel-Verdau verschiedener Substrate durch unbekannte Protease ............ 30 III.6.8. Elektrophorese und Gelfärbung ............................................................................ 32 III.6.8.1. SDS- Gelelektrophorese ................................................................................ 32 III.6.8.2. Silberfärbung ................................................................................................. 32 III.6.8.3. Zymographie ................................................................................................. 33 III.6.8.4. Glykoproteinfärbung ..................................................................................... 34 III.6.9. Nano LC-ESI-MS/MS, Q-TOF ............................................................................. 34 III.6.10. Reinigung des Latex von Euphorbia mauritanica L. ......................................... 35 III.6.10.1. Ionenaustauschchromatographie ................................................................. 35 III.6.10.2. Größenausschlusschromatographie ............................................................. 36 III.6.11. Charakterisierung der unbekannten Protease ...................................................... 37 III.6.11.1. Verhalten gegenüber pH-Wertveränderungen............................................. 37 III.6.11.2. Verhalten gegenüber Temperaturänderungen ............................................. 37 III.6.11.3. Verhalten gegenüber organischen Solventien ............................................. 37 III.6.12. Zellkultur ............................................................................................................. 38 III.6.12.1. Kultivierung von U-937 und THP-1 Zellen ................................................ 38 III III.6.12.1.1. Kryokonservierung ............................................................................... 38 III.6.12.1.2. Revitalisierung ..................................................................................... 39 III.6.12.2. Differenzierung mit rekombinantem humanen Interferon-gamma (rhINF-γ) ...................................................................................................................................... 39 III.6.12.3. Bestimmung der Zytotoxizität bzw. Zellviabilität mittels XTT .................. 39 III.6.13. Interleukin-6 ELISA ..........................................................................................
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