BCH3021 Lecture Notes - Lecture 21: Dapi, Peroxisome, Endothelium
Lecture 21 – Structure and Function of Peroxisomes and Lysosomes
• Prokaryotic cell
o Very little structure/organelles
• Evolution → eukaryotic cell
o Larger in size and 50% is occupied by organelles
Why do Eukaryotic Cells have Organelles?
• Overcomes increased volume to surface area
• Overcome diffusion limitations
• Allows increased reactant concentrations
• Provides containment deleterious pathways
• Allowed transition from extracellular digestion of food (prokaryotes) to
intracellular digestion (eukaryotes)
• Created vulnerabilities
o Reliance on other cells in organism, starvation in absence of nutrient
proximity
o Intracellular compartments provide niche for invading hostile
microorganisms
Visualisation
• Peroxisomes in endothelial cells
o Detected using antibody against a peroxisomal protein (Pex) (fixed cells)
o DAPI (blue fluorescence dye)
o Located next to mitochondria
Isolation of Lysosomes and Peroxisomes Subcellular Fractionation
• Can isolate by differential centrification
• Centrifuge at higher speeds and larger fragments will sink
• Can purify them
Properties of Peroxisomes
• Single membrane
• Small size, 0.1-1uM
• No genome
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• All proteins are nuclear encoded and imported, some ER, no relationship to
prokaryotic proteins
• Present in all eukaryotic cells (100-1000)
• Prevalent in liver and kidney
• Contains 15% mass of catalose
Peroxisome Function
• Contain diverse anabolic and catabolic pathways depending on cell type
o Make things and degrade things
• Catabolism
o Oxidation reactions for breakdown of a variety of substrates
o Important substrates: uric acid, amino acids, purines, fatty acids
o Xenobiotics – D-amino acids, alkanes, herbicides, methanol, phthalate
plasticisers, ethanol
o Catalysed by over 50 different enzymes – oxidase type
▪ R’H2 + O2 → R’ + H2O2
o Major site of oxygen consumption cell
o Reactive oxygen species (ROS)
▪ H2O2 is an ROS – important for signalling at low
concentrations but damaging at high concentrations
▪ Different sources of ROS: endogenous, antioxidant, exogenous
o Controlling hydrogen peroxide levels
▪ Catalytic mode: 2H2O2 → (catalase) O2 + 2H2O
▪ Peroxidation mode: R’H2 + H2O2 → (catalase) R’ + 2H2O
▪ E.g. methanol, ethanol, phenol, formaldehyde
▪ Step in degradation
• Anabolism
o Synthesis of complex lipids containing ‘ether linkages’
▪ Specific structures (=complex)
o Plasmalogens are essential component of myelin sheaths
▪ Resemble phosohplipids but with an ether linkage instead of
ester linkage
Using Endogenous Catalase to Visualise and Locate Peroxisomes in Cells and Tissues
• Using electron microscope
• Peroxisomes appear black in EM because so electron dense
• When oxidised – brown precipitate
o DAB (diaminobenzidine) is oxidised by catalase activity in tissue using
H2O2 → form elelectron dense precipitate (black blobs)
Β-oxidation of fatty acids occurs in Peroxisomes and Mitochondria
• Peroxisomes oxidise long chain and very long chain fatty acids
o 2C at a time to form acetyl CoA
• Acetyl CoA is exported for use by mitochondria in TCA cycle
• Mitochondria can only oxidise short chain fatty acids (<16c) – located in close
proximity
Peroxisome Biogenesis
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Lecture 21 structure and function of peroxisomes and lysosomes: prokaryotic cell, very little structure/organelles, evolution eukaryotic cell, larger in size and 50% is occupied by organelles. Visualisation: peroxisomes in endothelial cells, detected using antibody against a peroxisomal protein (pex) (fixed cells, dapi (blue fluorescence dye, located next to mitochondria. Isolation of lysosomes and peroxisomes subcellular fractionation: can isolate by differential centrification, centrifuge at higher speeds and larger fragments will sink, can purify them. H2o2 form elelectron dense precipitate (black blobs) Peroxisomes are removed from cell by autophagy: organelles have to be degraded, autophagosome double membrane. Inability to synthesise plasmalogens imperfect myelination of nerves. Lysosomal lumen contains 60 different soluble enzymes that promote intracellular. Hydrolases are delivered to lysosome lumen in an inactive state: how to stop, environment is acidic active at low ph (5, also expressed as pro-peptide, e. g. Cathepsin protease blocks active site and clips off peptide to become active.
