Chapter 3 - Part 2 Flashcards
What is facilitated diffusion?
Facilitated diffusion is a type of passive transport in which molecules go down a concentration gradient through a pore in the membrane created by a protein. Facilitated diffusion is necessary for molecules that are impermeable to the membrane (large, polar, or charged).
What is an ungated channel?
Ungated channels have no gates, and are therefore completely unregulated. Essentially, these are just holes in the membrane through which certain molecules can pass normally. responsible for maintaining resting membrane potential
What are the 5 major classes of non-enzymatic proteins?
-Structural Proteins
-Motor proteins
-Binding proteins
-Immunoglobins
-Biosignaling proteins
What is an enzyme linked receptor?
An enzyme linked receptor is a membrane receptor that displays catalytic activity in response to ligand binding. These enzyme linked membrane receptors have 3 primary domains: the membrane spanning domain, the ligand binding domain, and the catalytic domain. Enzyme linked receptors often lead to second messenger cascades.
Below is an Receptor Tyrosine Kinase (RTK) a common enzyme linked receptor. Monomer Dimerizes upon ligand binding. The dimer is the active form that phosphorylates additional cellular enzymes, including the receptor itself (autophosphorylation)
What is a G-protein coupled receptor (GPCR)?
GPCR’s are a large family of integral membrane proteins involved in signal transduction. They are characterized by their seven membrane spanning alpha helices.
How do GProtein Coupled Receptors exert their effects?
G-Protein Coupled Receptors are, as the name suggests, coupled to a heterotrimeric G protein. G proteins are named for their usage of GTP instead of ATP to power its actions.
When the appropriate ligand binds, the G protein’s affinity for the receptor increases. When the G protein binds to the receptor, it changes to the active state and can now activate nearby enzymes. Depending on the type of G protein, secondary messenger pathways will be activated or inhibited.
What are the three types of G proteins and what do they do?
- Gs stimulates adenylate cyclase, which increases levels of cAMP in the cell.
- Gi inhibits adenylate cyclase, which decreases levels of cAMP in the cell.
- Gq activates phospholipase C, which cleaves a phospholip from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3. IP3 can open calcium channels in the endoplasmic reticulum, increasing calcium levels in the cell
What are the three subunits of a G protein and what do they do?
- Alpha Sub Unit: Carries GDP/GTP to and from the G protein and the nearby enzymes to catalyze.
- Beta Sub Unit: Structural component of G protein
- Gamma Sub Unit: Structural component of G protein.
What is homogenization?
Homogenization is when a tissue of interest is crushed, ground, or blended into an evenly mixed solution. This is an important preliminary step for isolating and analyzing proteins in tissues.
What is centrifugation?
Centrifugation is the process of applying centrifugal force to a solution by spinning it. Centrifugation causes solid particles in the solution to collect at the bottom of the solution.
What are the two most common isolation techniques for proteins?
- Electrophoresis
- Chromatography
What is electrophoresis and what are the three main types?
Electrophoresis is the process of generating an electric field that separates proteins based on their charge and their size. Electrophoresis cells operate as electrolytic cells, meaning the cathode is negative and the anode is positive for all methods. ANALYTICAL
1. Native PAGE
2. SDS Page
3. Isoelectric Focusing
What is polyacrylamide gel?
Polyacrylamide gel is the standard medium for protein electrophoresis. This gel is a slightly porous mixture which solidifes at room temperature. The gel acts like a seive, allowing smaller, more charged particles to easily pass while slowing down larger, less charged particles.
Describe the factors (and equation) that determine the migration velocity of a protein?
Where E is the electric field, z is the net charge on the protein, and f is a frictional coefficient that is based on the protein’s size and weight. From this equation, it can be said that high electric fields in the gel (E), high charges on proteins (z) and small proteins (lower f) move fastest in polyacrylamide gel.
V = Ez/F
Describe Native PAGE?
Native PAGE is when you place proteins in a PAGE Gel and generate the electric field with no other modifications to the proteins. This means that proteins will migrate according to their size and charge.
This can be problematic, because a small protein with a low charge might end up moving about as fast as a big protein with a high charge.
However, this can be useful, because the proteins were not modified and therefore retain their function and can be recovered.
Native PAGE is best used on proteins that are known to be similar in either size or charge, so that the relative rates of velocity can be used to separate them based on their dissimilar characteristic (if the proteins have similar charge, then using Native PAGE helps determine the relative masses. Vice Versa.)
Describe SDS-PAGE (what two things does SDS do)
Sodium Dodecyl Sulfate (SDS) PAGE is useful because it separates proteins based only on their relative molar masses. This is accomplished through the addition of SDS to the proteins before running them on the gel. SDS serves two purposes. One, it disrupts noncovalent bonds in the proteins, denaturing them without breaking the primary structure. Two, it imparts on the proteins massive nonpolar groups that essentially mask the actual charge of the protein and instead leave it with a constant negative charge.
Because all proteins are carrying more or less the same negative charge, the relative velocities of migration are dependent entirely on the masses of the proteins.
What is Isoelectric Focusing?
Isoelectric Focusing is an electrophorytic method that exploits the basic and acidic properties of amino acids by seperating them on the basis of their isoelectric point.
Instead of a normal PAGE, the gel in the cell will have a pH gradient where the pH is low (positive charge) near the anode and the pH is high (negative charge) near the cathode.
At a pH below its pI, a protein will have a positive charge. This will cause it to migrate towards the cathode. However, as the protein moves towards the cathode, it will move into higher pH gel. Eventually, the gel’s pH will be equal to the protein’s pI. At this point the protein has no charge, and will move no longer.
Protein moves towards PI
Note, this does not allow us to separate proteins based on mass, only on pI.
What are the 4 most common forms of chromatography?
- Column Chromatography
- Ion Exchange Chromatography
- Size-Exclusion Chromatography
- Affinity Chromatography
