Comprehension - L05-L06 Flashcards
What is a facilitative transporter?
Binding of the solute triggers a conformational change in the transmembrane protein that exposes the solute to the other side
What is an example of a facilitative transporter?
GLUT4
Continued diffusion of glucose in the cell is possible because it’s phosphorylated
What are 5 examples of passive transport mechanisms?
- Diffusion through membrane
- Voltage gated ion channel
- Facilitative transporter
- Osmosis
- Ligand gated channel
What type of energy is used in active transport?
Hydrolysis of ATP (Primary active transport)
Flow of other substances down their concentration gradients (secondary active transport
What are the 3 types of primary active transport?
- P-type pump
- V-type pump
- ABC transport
How does the P-type pump work?
- Becomes phosphorylated during active transport
-Contributes to maintaining the membrane potential voltage in cells
What is pumped in/out per one ATP in the Na/K-ATPase P-type ion pump?
3 Na+ pumped out and 2 K+ pumped in
What are the steps of the Na+/K+ -ATPase P-Type ion pump?
- High affinity for Na+ and ATP is bound
- Protein closes once ions are bound
- Hydrolysis of ATP and pump is phosphorylated
- Release of ADP –> E2. Loses affinity for Na+, high affinity for K+
5-7. Dephosphorylation
7-8. ATP binds,–>E1. Low affinity for K+
What are the characteristics of V-type ion pumps?
Use ATP but don’t become phosphorylated
Transport H ions across organelles and vacuoles
Also found in PM of some cells
What are the characteristics of an ABC transporter?
-ATP Binding Cassette transporters
- Share similar structure of ATP binding domain
- Transport ions, lipids, peptides, and nucleosides
In secondary active transport, what is a symporter?
- Transports two substances in the same direction
- Also called cotransporter
In secondary active transport, what is an anti porter?
- Transports two substances in opposite directions
- Also called an exchanger
What does the Na+/glucose cotransporter do?
- Transport glucose from the lumen into epithelial cells
- Na+ ions [ ] is low inside cells
- Na+ ions moving down their [ ] gradient is used to drive the cotransport of glucose
What form of transport establishes the Na+ [ ] gradient in the first place?
Primary active P- Type
What is FRAP?
Fluorescence recovery after photobleaching
- Technique to study movement of membrane components
What are the 3 steps of FRAP?
- Label membrane w fluorescent dye
- Photobleach from a portion of cell
- Monitor reappearance of fluorescence in the previously bleached portion.
What does the rate of recovery of fluorescence measure in FRAP?
Rate of diffusion of the fluorescently labeled portion
When isolating membrane proteins, what is the first step? What happens in this step?
Lyse the cells!
Mechanical disruption (Hypotonic)
Pellet 1: insoluble membranes and proteins
Supernatant 1: soluble material
When isolating membrane proteins what is the second step? What happens in this step?
Isolate peripheral proteins!
High salt solution
Pellet 2: transmembrane proteins
Supernatant 2: Peripheral proteins
When isolating membrane proteins, what is the third step? What happens in this step?
Isolate Transmembrane proteins!
Strong detergent to break VDW interactions
Pellet 3: GPI-anchored proteins
Supernatant 3: Transmembrane protein
Where are GPI-anchored proteins usually found?
In detergent resistant portions of the membranes that are rich in cholesterol and sphingolipids
When isolating membrane proteins, what is the fourth step? What happens in this step?
Isolate GPI-anchored protein
Treatment w PI-PLC
Pellet 4: insoluble materials
Supernatant 4: GPI-anchored proteins
When performing SDS-Page, how are proteins separated by size?
Adding SDS to the samples. This is a negatively charged amphipathic detergent that gives proteins a uniformly negative charge and denatures the protein
What is the name of the dye that stains the proteins in polyacrylamide gel electrophoresis?
coomassie blue
