2_43 Flashcards
M) Urea, signal transd, P) IEX, how to find OUR/OTR (54 cards)
What is the urea cycle?
The urea cycle is the metabolic pathway that transforms ammonia to urea for
excretion from the body.
Briefly explain how the Urea cycle works and where.
How it works:
* Nitrogenous excretory products are removed from the body mainly in the urine.
* Ammonia, which is very toxic in humans, is converted to urea, which is nontoxic, very soluble, and readily excreted by the kidneys.
* The urea excreted each day by a healthy adult (about 30 g) accounts for about 90% of the nitrogenous excretory products.
* Urea is formed in the urea cycle from NH4+, CO2, and the nitrogen of aspartate.
Where it works:
* The cycle occurs mainly in the liver.
What are the substrates of the urea cycle?
NH3 (as derived from oxidative deamination of glutamate);
CO2;
aspartate;
three ATP.
What are the products of the urea cycle?
Urea; fumarate;
H2O.
What are important enzymes in the urea cycle?
- Carbamoyl phosphate synthetase I (Crazy People Sing)
- Ornithine transcarbamoylase (Opera Tunes)
- Argininosuccinate synthetase (After Singing)
- Argininosuccinate lyase (About Lying)
- Arginase (Again)
Urea cycle: What does the carbamoyl phosphate synthetase I do?
Converts ammonium and bicarbonate into carbamoyl phosphate.
This is the rate-limiting step in the urea cycle.
This reaction requires two ATP and occurs in the mitochondria.
Urea cycle: What does the Ornithine transcarbamoylase do?
Combines ornithine and carbamoyl phosphate to form citrulline.
Located in mitochondria.
Urea cycle: What does the Argininosuccinate synthetase do?
Condenses citrulline with aspartate to form arginosuccinate.
This reaction occurs in the cytosol and requires one ATP.
Urea cycle: What does Argininosuccinate lyase do?
Splits argininosuccinate into arginine and fumarate.
Occurs in the cytosol.
Urea cycle: What does Arginase do?
Cleaves arginine into one molecule of urea and ornithine in the
cytosol. The ornithine is then transported back into the mitochondria for entry
back into the cycle.
What are the types of intracellular signalling molecules?
1) Adaptor protein
2) Amplifier/Transducer
3) Bifurcation proteins
4) Integrator proteins
5) Latent gene regulatory proteins
6) Messenger proteins
7) Modulator proteins
8) Relay protein
9) Scaffold
What are scaffold proteins? What are the advantages of scaffolding?
- Scaffold proteins organize groups of signalling proteins into signalling complexes.
- They also hold proteins at a specific location.
- This is advantageous so that the signal can be relayed with precision, speed, efficiency and specificity.
What are characteristics of GPCRs (G-protein coupled receptors)?
- Metabotropic (meaning they initiate intracellular metabolic signalling cascades), there is no direct channel opening
- Largest family of cell-surface receptors
- Have 7-transmembrane domains which bind to a ligand to activate a G-protein
- When GTP binds, the activated G-protein dissociates to an alpha and beta-gamma subunit
- These subunits can activate an ion channel or a membrane enzyme e.g. adenylyl cyclase
What are main G-proteins?
- Gs (activates adenylyl cyclase, increasing cAMP)
- Gi (inhibits adenylyl cyclase, decreasing cAMP)
- Gq (activates phospholipase C, increasing intracellular calcium)
GPCRs: Signalling mechanisms of Gs branch of trimeric G-proteins?
- Signal molecule binds to GPCR causing its activation. This activates the adenylyl cyclase, which activates the alpha subunit of the Gs protein
- This activation increases cAMP concentration in the cytosol, PKA subunits are activated. PKA enters the nucleus through the nuclear pore, where they phosphorylate transcription regulatory protein CREB
- Phosphorylated CREB recruits coactivator CBP, stimulating gene transcription through activation of the target gene
IEX: Define pI (isoelectric point) and pH
- pH: A measure of how acidic or basic a solution is; based on hydrogen ion concentration [H+].
- pI (isoelectric point): The pH at which a molecule (usually a protein) has no net charge.
GPCRs: Signalling mechanisms of Gq branch of trimeric G-proteins?
1) PLC is a membrane-bound enzyme, which is activated by the Gq protein once a signal molecule binds
2) IP3 binds to IP3 sensitive Ca2+ channel, causing release of Ca2+ from lumen of the ER
3) The Ca2+ ions join PKC (protein kinase C) which then phosphorylates substrates
What is IEX?
- Ion Exchange Chromatography (IEX) is a separation technique that separates molecules based on their net charge.
- It works by using a stationary phase (column) with charged functional groups that interact with oppositely charged molecules in the sample.
- This interaction allows for the separation and purification of charged biomolecules like proteins, peptides, and nucleic acids
IEX: What are the 3 key things to be aware of in terms of pH and pI?
1) Know your proteins pI: The pI is the pH at which your protein has no net charge, so it won’t bind to cation or anion exchange resins as its neutral
2) Set the pH appropriately:
* If the protein has to bind to cation exchanger (pH less than pI, protein is positive),
* if the protein binds to anion exchanger (pH greater than pI, protein is negative),
* if neither (for flow-through) then pH=pI, the protein is neutral
If the pH is too close to pI: protein might not bind properly, elute prematurely
If the pH is too extreme, the protein could denature/precipitate
3) Best practice: use a buffer pH at least 0.5/1 unit above/below the protein’s pI
What are the conditions for a molecule to bind to a cation or anion exchanger?
- At pH less than pI: Molecule is positively charged → binds to cation exchanger (negative resin).
- At pH greater than pI: Molecule is negatively charged → binds to anion exchanger (positive resin).
- At pH = pI: Molecule is neutral → does not bind to ion exchange resin.
IEX: How can you separate proteins from each other?
- By altering the buffer conditions (pH, salt concentration), proteins with different net charges can be selectively eluted and separated.
- The separation relies on the interaction between charged proteins and a charged SP (resin) within the chromatography column.
IEX: why use a anionic buffer? Give an example
- Cation exchange: a buffer with an anionic counterion (e.g., chloride, acetate)
- Why: The resin is negatively charged and binds positively charged proteins. Anionic buffers help maintain ionic strength and charge neutrality without displacing the target protein.
Eample: Acetate buffer (pH 6.0)
IEX: why use a cationic buffer? Give an example
- Anion exchange: a buffer with a cationic counterion (e.g., ammonium, Tris)
- Why: The resin is positively charged and binds negatively charged proteins. A cationic buffer balances the charge in the mobile phase without competing with the protein for binding.
Example: Tris-HCl buffer (pH 8.0)
Signal transduction: upon what factors does the binding of ligand-receptor depend on?
- type of ligand with type of receptor
- cell type
- location of receptor in cell
- downstream signaling complexes that are linked
