Breaking to Reveal: The Peroxisome Flashcards
Lecture 10
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
What is the mitochondria’s density?
1.18 g/cm^3
What is the peroxisome’s density?
1.23 g/cm^3
What are the three potential methods for determining the contents of a cell fraction?
- Microscopic observation
- Enzymatic assays for marker enzymes
- Probing for marker molecules (usually proteins, but non-enzymes)
During an enzymatic assay, which enzyme(s) was associated with the lysosome? Where was the fraction found?
Acid phosphatase, found at the top of the tube
During an enzymatic assay, which enzyme(s) was associated with the mitochondria? Where was the fraction found?
Cytochrome oxidase, found near bottom of the tube
Researchers believed there may have been some spill over from other organelles into the mitochondria’s fraction after finding the cytochrome oxidase. What did they do to confirm they had found the mitochondria’s fraction? What did they discover?
Labeled mitochondrial DNA with 14C. They discovered they had accurately identified the mitochondria because the peak for the DNA was in the same place as the peak for the marker enzyme.
During an enzymatic assay, which enzyme(s) was associated with the peroxisome? Where was the fraction found?
Catalase and ureoxidase
How was the peroxisome discovered?
During the enzymatic assays after isopycnic centrifugation, ureoxidase was found in the fraction where the lysosome was. This was confusing because the lysosome’s pH was too low for this enzyme to be active there. This indicated the ureoxidase was operating in another organelle.
What does SDS do during SDS-PAGE (SDS-Polyacrylamide Gel Electrophoreses)?
Covers the polypeptides, ensuring
1. a uniform charge to mass ratio (imparts negative charge on all protein samples)
2. uniform shape by denaturing polypeptides
After this, the only physical difference between polypeptides is molecular weight.
What does SDS-PAGE do?
Ensures the only difference between polypeptides is molecular weight, so they can be separated based on size
Describe the process of SDS-PAGE
- Denature samples with SDS.
- Place mix of proteins on gel and apply the electric field, causing proteins to move.
- Stain the gel to visualize the separated bands.
Which side of the gel are the larger proteins are in SDS-PAGE?
They are closer to the negative pole. They didn’t move as quickly as smaller particles through the existing holes.
What is immnoblotting (Western blotting)?
method for detecting marker proteins in cell fractions after SDS-PAGE, reveals which fraction target organelles are in
Describe the process of immunoblotting.
- Take the SDS poly gel after SDS-PAGE.
- Apply electric field from the side, moving the proteins out of the gel.
- To catch proteins, put membrane behind the gel and allow polypeptide to bind to it.
- Add an antibody to find the target protein on the membrane.
- Add a secondary antibody with a detectable (fluorescent) molecule to attach to the first antibody
What kind of cells have peroxisomes?
virtually all eukaryotic cells
What is the diameter of the peroxisome?
between 0.1 and 1 micrometer
What is the diameter of the peroxisome based on?
physiological state and growth conditions of the cell
How many peroxisomes are inside a cell?
varies
Are peroxisomes membrane-bounded or not?
Membrane-bounded
What enzymes are present in peroxisomes?
All peroxisomes have catalases.
Some oxidases are in peroxisomes.
Peroxisomes may have a crystalline form? Under what conditions does it form?
Non-primate cells that have a high concentration of ureoxidase crystallize.
Describe peroxisome’s relationship to O2 utilization.
The peroxisome is the first organism that utilized atmospheric oxygen when O2 levels rose on earth. The peroxisome reduced O2 to protect cells from oxidative damage.
Why do people believe the peroxisome is less important now?
After the evolution of the mitochondria, the mitochondria began reducing O2 and coupled the redox reaction to ATP production, which the peroxisome could not do.
What are the five functions of the peroxisome?
- Hydrogen peroxide metabolism
- Catabolism of unusual molecules
- Metabolism of nitrogen-containing compounds
- Detoxification of organic compounds and reactive oxygen species (radicals)
- Beta-oxidation of fatty acids
Which enzyme forms hydrogen peroxide? When does that occur?
Oxidase forms hydrogen peroxide as a byproduct when utilizing O2.
Which enzyme metabolizes hydrogen peroxide?
Catalase in the peroxisome metabolizes hydrogen peroxide into water and O2.
Which unusual molecules are catabolized by the peroxisome?
D-amino acids
Xenobiotics
If a compound contains nitrogen, what will likely happen to it?
Will be metabolized by the peroxisome
Which kinds of molecules are detoxified by the peroxisome?
Organic compounds (ex. aldehydes, ethanol) and reactive oxygen species (hydrogen peroxide, radicals)
Why is it necessary for the peroxisome to destroy reactive oxygen species?
to prevent oxidative stress
Which enzyme is responsible for detoxifying organic compounds?
catalase
What does the beta-oxidation of fatty acids do?
Shortens long fatty acid chains over time and produces acetyl CoA
What happens to the acetyl CoA that is produced after the beta-oxidation of fatty acids in the peroxisome?
either anabolism or used in the TCA cycle as energy (building block of lipids)
