Breaking to Reveal: The Peroxisome
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What does microscopy reveal about organelles? What can’t it reveal?
Reveals location, size, and shape, but can’t reveal function
What is cell fractionation?
Process to break apart cells and isolate different organelles; bridges microscopy and biochemistry
What is differential centrifugation?
a key technique for separating organelles based on mass, shape, size, and density
During differential centrifugation, what is the supernatant?
the part of the test tube where the organelle of interest is after centrifugation; composed of lighter cell materials
During differential centrifugation, what is the pellet?
the bottom of the test tube after G-force; composed of denser cell materials
What is the cytosol?
the soluble part of the cytoplasm; remains of a cell after very high speeds
What is the cytoplasm?
substance between the cell’s plasma membrane and the nucleus
What is the process of differential centrifugation?
• Filter homogenate (disrupted cells without clumps) to remove clumps of unbroken cells, connective tissue, etc. • Pour out the supernatant, then spin it at higher speeds.
What is centrifugation at low speeds called? What speed? What does it separate out?
Clinical centrifugation spins at 600 g x 10 min and filters the nuclei into the pellet.
What is centrifugation at medium speeds called? What speed? What does it separate out?
Super centrifugation occurs at 15,000 g x 5 min and filters out the mitochondria, chloroplasts, lysosomes, and peroxisomes.
What is centrifugation at high speeds called? What speed? What does it separate out?
Ultraspeed centrifuges spin at 100,000 g x 60 min and separate the plasma membrane, microsomal fraction (fragments of the ER), and large polyribosomes.
What is centrifugation at very high speeds called? What speed? What does it separate out?
Ultraspeed centrifuges spin at 300,000 g x 2 h and separate ribosomal subunits, small polyribosomes, and eventually leave behind the cytosol.
DNA and RNA have similar S values. Which is more dense?
RNA is more dense, at about 2.0 g/cm^3 compared to 1.7 g/cm^3.
What do the relative sedimentation values communicate about centrifugation?
Very different sedimentation values indicate there’s a good chance of separating organelles via centrifugation. Harder to separate them with similar S values.
Peroxisomes and mitochondria have the same S-value. Which is more dense?
Peroxisomes are more dense.
What does differential centrifugation separate based on?
mass, shape, size, and density
What does rate zonal centrifugation separate based on?
S-value (mass and size)
What does equilibrium (buoyant) density gradient centrifugation separate based on?
density
What does sucrose do in rate-zonal and equilibrium density gradient centrifugation?
Creates a barrier for molecules separated in the centrifuge’s field based on S-value (for rate zonal) and density (for buoyancy)
What happens to molecules closest to the bottom of the test tube in rate zonal centrifugation? Why?
They are slowed down due to sucrose’s resistance. There’s a higher sucrose concentration at the bottom of the tube.
Which forms of centrifugation have a solute gradient?
rate zonal and equilibrium density gradient centrifugation
What are the 3 names for the form of centrifugation that separates based on density?
Equilibrium Density Gradient Centrifugation Buoyant Density Centrifugation Isopycnic Separation
Why does isopycnic centrifugation separate based on density?
The range of concentration of the sucrose in the tube allows organelles to migrate to an equivalent point of density.
What is the density of the lysosome?
1.12 g/cm^3