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Isolation of Subcellular Fractions from Tissues and Cellular Sources

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Isolation of Subcellular Fractions from Tissues and Cellular Sources DIPLOMARBEIT Isolation of subcellular fractions from tissues and cellular sources Zur Erlangung des akademischen Grades einer Magistra der Pharmazie an der naturwissenschaftlichen Fakultät der Karl-Franzens Universität vorgelegt von Nicole Penkoff begutachtet von O. Univ.-Prof. Dr. Bernhard-Michael Mayer Institut für Pharmazeutische Wissenschaften April, 2014 DANKSAGUNG Mein Dank gilt O. Univ. Prof. Dr. Bernhard-Michael Mayer für die Möglichkeit an diesem Institut eine Diplomarbeit zu schreiben und damit einen guten Einblick in die Forschung zu bekommen. Ganz herzlich möchte ich mich bei Dr. Astrid Schrammel-Gorren für die tolle Betreuung, humorvolle Diskussionsbereitschaft und Ermutigung während der gesamten Diplomarbeit bedanken. Vielen Dank für die lehrreiche und zugleich lustige Zeit! Weiterhin danke ich Dr. Karoline Pail für die gute Einführung in das „Western Blotten“ zu Beginn meiner Diplomarbeit und für die vielseitigen Diskussionen. Vielen Dank an Dr. Alexander Kollau für die Erläuterungen und Diskussionen über Enzyme, Mitochondrien und zuallerletzt schließlich auch Koronararterien. Für die Unterstützung während meines kleinen Ausflugs in die Zellkultur danke ich Kerstin Geckl vielmals. Bei Ines Neubacher möchte ich mich für das Gießen einiger Gele, ihre guten Tipps und für die Durchführung der Bestellungen bedanken. Außerdem danke ich allen anderen Mitarbeitern dieses Instituts, die mir bei Unklarheiten mit Rat und Tat zur Seite standen und mir durch die freundliche Arbeitsatmosphäre die Laborzeit zum Vergnügen machten. Schließlich möchte ich mich noch bei meiner Familie für die Ermöglichung dieses Studiums bedanken, sowie bei meinen Freunden, für ihre Unterstützung und Begleitung. 2 Meinen Brüdern René und Marcel in Liebe gewidmet. 3 Table of contents 1. Introduction ......................................................................................................... 7 1.1. Subcellular structures and marker enzymes ................................................ 7 1.1.1. Plasma membrane ................................................................................. 7 1.1.2. Cytosol ................................................................................................... 8 1.1.3. Mitochondria ........................................................................................ 10 1.1.4. Nucleus ................................................................................................ 12 1.1.5. Golgi apparatus and endoplasmatic reticulum ..................................... 13 1.2. Objectives .................................................................................................. 14 1.2.1. Role of eNOS in ATGL deficiency ........................................................ 14 1.2.2. Vascular aldehyde dehydrogenase 2 expression ................................ 16 2. Methods............................................................................................................ 18 2.1. Materials .................................................................................................... 18 2.2. Preparation of mouse tissue (lung, liver) and porcine coronary arteries ........................................................................................ 22 2.2.1. Homogenization ................................................................................... 22 2.2.2. Isolation of subcellular fractions by differential centrifugation .............. 23 2.2.3. Sucrose density gradient centrifugation ............................................... 25 2.2.4. Purification by aqueous dextran/PEG two-phase system .................... 27 2.2.5. Isolation and purification of liver nuclei by sucrose enrichment ........... 29 2.3. PAECs ....................................................................................................... 31 2.3.1. Cell culture ........................................................................................... 31 2.3.2. Homogenization methods .................................................................... 31 2.4. Measurement of enzyme activities in murine tissues ................................. 33 2.4.1. Measurement of LDH activity in lung and hepatic cytosol .................... 33 2.4.2. Cytochrome c oxidase activity in lung and hepatic mitochondria ......... 34 4 2.5. Immunoblotting ........................................................................................... 35 2.5.1. BCA assay ........................................................................................... 35 2.5.2. Gel electrophoresis and Western blot .................................................. 35 2.6. Statistical analysis ...................................................................................... 37 3. Results ............................................................................................................. 38 3.1. Murine lung (wild type) ............................................................................... 38 3.1.1. Distribution of eNOS in unpurified subcellular fractions ....................... 38 3.2. Protein expression in lung preparations of wild type and ATGL (-/-) mice ......................................................................................... 38 3.2.1. Marker enzyme expression .................................................................. 38 3.2.2. LDH and cytochrome c oxidase activities ............................................ 40 3.2.3. eNOS protein expression ..................................................................... 41 3.3. Murine liver (wild type) ............................................................................... 42 3.3.1. Distribution of eNOS in unpurified subcellular fractions ....................... 42 3.3.2. Purification of murine liver fractions ..................................................... 43 3.4. Protein expression in liver preparations of wild type and ATGL (-/-) mice .......................................................................................... 47 3.4.1. Marker enzyme expression .................................................................. 47 3.4.2. Lactate dehydrogenase and cytochrome c oxidase activity ................. 50 3.4.3. eNOS protein expression ..................................................................... 50 3.5. Porcine coronary arteries ........................................................................... 52 3.5.1. Effects on mitochondrial integrity by different homogenization methods ............................................................................................... 52 3.5.2. Distribution of eNOS and ALDH2 in unpurified subcellular fractions .... 53 3.5.3. Purification of light membrane fraction ................................................. 55 4. Discussion ........................................................................................................ 56 5. Summary .......................................................................................................... 60 5 6. Zusammenfassung ........................................................................................... 60 7. Abbrevations .................................................................................................... 61 8. References ....................................................................................................... 64 6 1. Introduction 1.1. Subcellular structures and marker enzymes Cells are the smallest independent functional units of the organism. As shown in Fig. 1, the eukaryotic cell is mainly composed of plasma membrane, cytoplasm, mitochondria, Golgi apparatus, smooth and rough endoplasmatic reticulum (ER), peroxisomes, lysosomes, and nucleus [1]. Figure 1: Structure of an eukaryotic cell; Mutschler, E., Schaible, H-G., Vaupel, P., Anatomie Physiologie Pathophysiologie des Menschen, Wissenschaftliche Verlagsgesellschaft, 2007. Marker proteins are more or less exclusively located in/on the respective organelles and detection of such markers is routinely used in biochemical studies to characterize plasmalemma and cell organelles [2]. Relevant subcellular structures for this work are described in the following chapters. 1.1.1. Plasmalemma Plasmas membranes are biomembranes that constitute cell boundaries and consist of a hydrophobic matrix, formed by an oriented double layer of phospholipids to which proteins are bound in different forms [3]. Singer and 7 Nicolson (1972) presented the organization of proteins and lipids of biological membranes as fluid mosaic model [4], which is still relevant today to understand structure, function, and dynamics of biological membranes [5]. In this model, integral membrane proteins are a heterogeneous set of globular molecules, each arranged in an amphipathic structure. The ionic and highly polar groups protrude from the membrane into the aqueous phase, and the nonpolar groups largely point to the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipids, which is largely organized as a discontinuous fluid bilayer [4]. Important biochemical processes essential for cell homeostasis occur within the cell membranes [3], including transport of molecules in and out of the cell, intercellular signaling, and cell adhesion [5,6]. Microvilli anchored to the plasma membrane increase the surface area for absorption of nutrients from surrounding medium [6]. Na+/K+-ATPase found in cells of all higher eukaryotes is a plasmalemma-located transmembrane ATP-dependent ion pump.
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