Cell Fractionation

What method can be used to determine the shape, size, and location of organelles?

Microscopy

What is one method used to understand how cells function?

Cell Fractionation

What is Cell Fractionation?

The process of methodically disrupting cells and collecting specific organelles to study in isolation in order to run specific tests to figure out function

What are important components of Cell Fractionation?

1. Choice of material → use cells that have a lot of the organelle of interest (wouldn’t use liver cells to study chloroplasts)

2. Method of disruption → can put cells in blender to break apart tissue and membrane

3. Vehicle of disruption → environment that the disrupted cells are put in; need:

   • buffered solution to avoid pH changes

   • proper ions around (cofactors)

   • osmotic stabilizers to prevent lysis of organelles

   • protein stabilizers and protease inhibitors

   • reducing agents (since cytoplasm is reduced environment)

4. Method of separation → most commonly used method is centrifugation

   • Differential Centrifugation

   • Rate-Zonal Centrifugation

   • Equilibrium Density Gradient Centrifugation

5. Method of identification → determine which organelles are which

   • Microscopic Observation (not very useful)

   • Enzymatic Assays for Marker Enzymes

   • Probing for Marker Molecules

What is Differential Centrifugation?

Used when objects of interest have a very large difference in S values; disrupt cells in blender, then run that through a filter to get the filtered homogenate; then centrifuge the filtered homogenate, and objects will separate based on mass, shape, size, and density; start at low speed, then pour supernatant into new tube and centrifuge that and so on; larger organelles will precipitate out at lower speeds

How is the cytosol different than the cytoplasm?

The cytoplasm is the liquid and large objects inside of a cell; the cytosol is the soluble part of the cytoplasm (just the liquid)

What will be in the pellet when doing clinical speed differential centrifugation?

Large objects like the nucleus

What will be in the pellet when doing super-speed differential centrifugation?

Large and dense organelles like mitochondria, chloroplasts, lysosomes, and peroxisomes

What will be in the pellet when doing ultra-speed differential centrifugation?

Plasma membranes and microsomes (ER), ribosomes, and eventually the cytosol (last thing left in supernatant)

What is a Sedimentation Coefficient?

It is the ability of an object to sediment when centrifuged; takes into account mass, shape, size, and density; unit is the Svedberg Unit; larger S value means object will sediment out quicker

What is Rate-Zonal (Density Gradient) Centrifugation?

Used for organelles of similar densities, but only slightly different S values (ex. mitochondria and lysosomes); this technique accentuates differences in S values between objects; fill tube with dense solution (sucrose); there is a lower concentration of sucrose at the top of the tube than the bottom, so the top of the tube is less dense than the bottom; place sample in tube and centrifuge it; sample will separate based on S value, in which mass and size are most significant; organelles with larger S values will move farther; creates tight bands of organelles that are distinct from each other; to get it out of the tube, you poke a hole in the bottom and drip the solution out into collecting tubes

What is Equilibrium (Buoyant) Density Gradient Centrifugation?

Used for objects that have the same S value, but different densities (ex. mitochondria and peroxisomes); works exactly the same as Rate-Zonal, but the dense solution (sucrose) used is much denser; as the organelles move through the tube, they will stop when the density of the solution is equal to their own; organelles separate based on density

What are enzymatic assays for marker enzymes?

de Duve figured this out; it is a way to determine the contents of cell fractions after centrifugation; it tracks enzymes that are unique to each organelle; the plots show the activity of certain enzymes against the density of the fractions

How did enzymatic assays lead to the discovery of peroxisomes?

When de Duve did an enzymatic assay and was looking at the results, he found that both acid phosphatase (marker for lysosomes) and urate oxidase were found in the same fraction, but this was puzzling because urate oxidase doesn’t function at low pH (lysosomes are acidic); de Duve concluded that there was another organelle mixed into the lysosome, which led him to try and discover what this other organelle was, and through running subsequent experiments, he was able to determine the identity of this new organelle, which was a peroxisome

What are peroxisomes?

• They are found in almost all eukaryotic cells

• They are about 0.1-1 μm in diameter

• Not all cells have the same number of peroxisomes

• They are membrane-bound

• They contain catalases and oxidases (oxidases aren’t in human peroxisomes, but they are in other mammals)

• They are very dense and have a crystalline catalase core

• They are a major site of O2 utilization; they are thought to be very ancient and were originally purposed for detoxifying atmospheric O2, but when mitochondria came into play, they kind of overshadowed peroxisomes in terms of O2 usage

What is probing for marker molecules?

It is a technique to determine the contents of cell fractions; it typically looks for proteins (enzymes) and lipids; when it looks at these molecules, it only looks at whether or not they are present, not their activity like with enzymatic assays; this technique uses SDS-PAGE and immunoblotting

What is SDS-PAGE?

A technique used to identify cell fractions; first you run the fraction through polyacrylamide gel, which has an electric charge running through it; to ensure the fraction separates based on molecular weight and not charge, you mix the fraction with an ionic detergent, SDS, which is very negatively charged and uniformly coats the protein in negative charge and also denatures the proteins, giving the polypeptides a uniform charge to mass ratio and a uniform shape; the smaller polypeptides then move further and faster down the gel than the larger ones; staining can help people see the polypeptides in the gel, but you can’t really tell which one is which

What is immunoblotting?

A technique that allows us to find a specific polypeptide on a gel; you take your gel from SDS-PAGE and press it against a thin membrane, then apply an electric field perpendicular, causing the polypeptides to transfer to the membrane; then add an antibody that is specific to the polypeptide of interest, and it will bind to the polypeptide; then add secondary antibody with a marker attached to it and it binds to the primary antibody, making the polypeptide visible under a scanner; this is like indirect immunofluorescence

What are the functions of peroxisomes?

• Hydrogen Peroxide Metabolism → oxidizing organic molecules into hydrogen peroxide using O2; the H2O2 is then rapidly converted by catalase into H2O and O2

• Catabolism of unusual molecules → the peroxisome breaks down molecules like D-amino acids and xenobiotics (molecules that are not naturally occurring within a cell, like alkanes)

• Metabolism of Nitrogen-containing compounds

• Detoxification

  • Organic compounds → breaks them down using H2O2 and catalase

  • Reactive oxygen species → converts reactive species like H2O2, OH-, and O₂^- into non-reactive species; this reduces oxidative stress on the cell

• Beta-oxidation of fatty acids → removes the reduced carbon tails of fatty acids and converts them into Acetyl CoA, which can be used for anabolic reactions and the Krebs Cycle

  • in humans, this process begins in the peroxisomes but ends in the mitochondria

Why is it important that hydrogen peroxide metabolism happens in peroxisomes and not the cytosol?

Because H2O2 is very reactive and can be damaging to cells, so the compartmentalization of the reaction happening in the peroxisome is important

What is a Dalton?

It is used to measure the weight of proteins; it is equivalent to about 1/12 of the weight of a Carbon atom, or 1 Hydrogen atom

Can you use cell fractionation to determine the exact, precise location of a protein or its function?

No, cell fractionation cannot tell us the exact location of a protein within an organelle, it can only tell us it is somehow associated with that organelle, and it cannot tell us the function of a protein; for example, it cannot definitively determine if a protein is a plasma membrane protein, only that it happened to appear in the same fraction as the plasma membrane, so there is some sort of association between the two

What part of the protein’s structure does the SDS detergent disrupt?

The tertiary and secondary structure; it disrupts all non-covalent bonds, but not the covalent bonds

Is it necessary to do cell fractionation for all centrifugation?

No, cell fractionation is only used when you want to isolate a specific organelle, centrifugation can be done without destroying the cell