How do proteins control entry into cells? Oct 1 and 3

1. How do proteins control entry into cells?Oct 1 and 3 Cell Bio Term Paper Titles Due Today 5points-email Dr Wilson for proposal approval and lets find a good project Regulation of membrane transport: How and Why? • What is the difference between diffusion and osmosis? • What is solute flux? • How can you tell if diffusion of a solute is occurring via simple diffusion or if diffusion requires a transmembrane protein? • Active transport mechanisms cover when gradients are not enough. • Secondary Active uses ATP to generate a gradient that provides the actual Delta G. • Using proton gradients to generate Delta G and ATP • Calculating Delta G for membrane transport.

2. 10 Repeats Assignment for Wed Oct 3rd: 10 points • Glycolysis and Gluconeogenesis: Write using pen or pencil the names of all enzymes, reactants, and products required for one glucose to be oxidized to two molecules of pyruvate in the cytosol (glycolysis). Show the additional enzymes required for two pyruvates to be converted back into a single glucose (gluconeogenesis). Show the enzymes used to create two lactic acid molecules in cells lacking mitochondria or in cells that are hypoxic. Write this same thing out (by hand) and repeat this nine more times (10 times total).

3. Practice Quiz 1) Draw a phospholipid and indicate what portions are hydrophobic and hydrophillic. 2) To maintain membrane fluidity, how might the length and saturation of PL fatty acid tails in plasma membranes change if a warm water organism moved into cold water? 3) Why in terms of its lipid is cod (fish that lives in very cold water) good for you? Why does this fish have these lipids? 4) Which PL flip flop is more common? Why? Lateral Transverse Diverse Political 5) Draw and label a cis and trans -HC=CH- double bond with respect to the orientation of its hydrocarbons 6) True/False: Cholesterol is important for reducing the fluidity of all plant plasma membranes. 7)What functional groups anchors most transmembrane proteins to the intra and extracellular sides? 8) Draw a plasma membrane with at least 10 different features shown and labeled. 9) Describe two ways to link a carbohydrate to a membrane protein.

4. How do red blood cells function? What are the cellular Problems/Solutions that must be overcome? RBC HAVE NO ORGANELLES or DNA! Ability to anchor materials to RBC membrane? Glycosylation- Protein +/- charged ends- Ability to for RBCs to be recognized in body as native/foreign? Glycolipids: ABO- Glycoproteins: Other antigens- Ability to be excluded from filtration by kidney? (-)Charge on RBC/ fenestra: Like repels Like=> Ability to have large surface area and low friction in capillary? How does RBC shape address this- Glycophorin: is the classic well studied RBC integral membrane protein ABC subtypes- Sub-type A Relation to Plasmodium and Influenza- What happens if you don’t have genes for glycophorin A? How can you prevent malarial infection by modifying A?

5. Glycophorin is an important membrane in red blood cells that provides some of our (non-ABO) blood types. Positive hydropathy indexes in the sequence indicated non-polar amino acids. How many transmembrane sequences are present?

6. What amino acids would you expect at the locations where the glycosylations occur on glycophorin? Why is the carbohydrate sialic acid (-charged) important for the extracellular side on an RBC?

7. How does Osmosis and Diffusion differ? • What is a semipermeable membrane? • What is Diffusion? • How does a Concentration Gradient create a Delta G gradient? • When is chemical equilibrium reached across a membrane? • What factors determine how much diffusion occurs over an area • 1 Solute Size: • 2 Solute Polarity: • 3 Solute Ionic Charge: • 4 Membrane Characteristics: • Osmosis :movement of water down its concentration gradient.Osmosis occurs when solutes cannot move but water can! Hence water moves to dilute the solute on the more concentrated side of a membrane. • Osmosis stops when solute on both sides is equally dilute.

8. How do we measure the transport rate for a solute as it moves across a membrane? (This is “Solute Flux”) • Considerations: • Area diffusion can occur on? • Concentration difference? • Permeability? • Mathematical Formula: (flux/area)(Diffusion Constant)(Conc. Difference)=Flux per area • Rate of inward flux=permeability coefficient X conc. Diff =Moles/second-cm2 =flux

9. Non-polar materials like carbon dioxide and oxygen easily diffuse through the phospholipid bilayer. Intra or extracellular enzymes may then change the molecule to fix its location in a cell. Example: Carbonic Anhydrase How much CO2 Moves? This is called “Flux”

10. Flux rates for non-protein mediated transport follow strictly linear relationship.IF IT LOOKS HYPERBOLIC IT IS ENZYMATIC! Sometimes proteins are required for materials to pass down a concentration gradient through a membraneIf you observe that flux can be saturated by increased concentrations, you know that Facilitated Diffusion is responsible for the transport. Two Facilitated Diffusion Choices: Carrier Mediated or Channel Mediated

11. Facilitated Transport implies that a protein (pore/carrier) is involved, hence a Km and Vmax can be measured. The Classic Example of Fascilited Transport of Glucose: • Consider what happens to glucose reabsorption and the epithelial cells that line the nephron during hypergycemia (diabetes)! • Why do you want to reabsorb glucose? • Enzymes pull glucose and return it to your blood! • Glucose transport has a Vmax and can be saturated! • When with respect to Km and Vmax will glucose appear in urine and when/why will the person become dehydrated (die)? Urine [Glucose] Vmax Passed! • I V • I • I • I • I • I • L________________________________ • Blood [glucose]

12. Carrier Proteins move materials across the hydrophobic center of a plasma membrane! What are their Requirements???? 1) Why is a Concentration Gradient REQUIRED! • Provides the delta G 2) Why is a Conformation change REQUIRED! • Flexes to permit passage (“door effect” 3) Why is Substrate Specificity REQUIRED! • “Door” (active site/channel) only opens to specific “key” (substrate) • MM-Enzyme Kinetics! V= Vm(deltaS)/Km +(delta S) • Competitive Inhibition is possible! Transport enzymes can be saturated! • What are the Three Carrier Types: • 1) Uniport Carrier- • 2) Antiport Carrier- • 3) Symport Carrier- • These are three are DIFFERENT from Pores which are basically holes that are present and do not require conformational changes to operate. • Pores:no directionality as long as a Concentration Gradient exists!

13. The glucose transporter (GLUT) of the red blood cell is a very well studied example of a uniport carrier! A Simple Glucose Concentration Gradients all that is Required!

14. The chloride/bicarbonate Exchange (antiport) carrier of the red blood cell is a well studied example of an antiport! Remember: Concentration gradient for Cl or HCO3 is required! Carbonic Anhydrase converts substrates: Keeps a gradient for HCO3

15. Symport Carrier is a classic way to move glucose in the intestinal epithelium at the same time…..ONLY IF A CONENTRATION GRADIENT EXISTS FOR GLUCOSE! This is how Gatorade works!This is also used to save babies with diarrhea! • How does this work?

16. There are also channel proteins that permit the free passage of solutes through a hole, pore, or channel! • Names: Ion Channels, Porins, Permeases and Aquaporins! • Important Ions: Na+, K+, Ca++ and Cl- • Trick: use a conformation change to open/close the hole! • One renal aquaporin molecule allows 3 billion H2O into a cell/second! • How are they regulated regarding open/closed state? • Aquaporin: add to notes!  • Ligand-gated channels: • Voltage-gated channels: • Mechanosensitive channels: • Remember: porins in prokaryotes have larger diameters and lower substrate specificities than in eukaryotes.

17. Active Transport Proteins (pumps) require the presence of free energy (from ATP) for their transport activity to function. They push solutes against large concentration gradients! • Where do they get the free energy? • What happens relative to the concentration gradient? • Direct Active Transport: ATP is energy source! • We call these “ATP-ase pumps” • Indirect Active Transport: Energy released from chemical gradient for A, is used to move B-against its gradient! • These can use “Symport or Antiport” actions! • Directionality: pump only goes in only one way!

18. There are four classes of ATPase Pumps! • 1) P-type ATPase: classic of eukaryote PM • ATP+Enzyme ADP + Enzyme-Pi  Enzyme + Pi + Energy + Pumping 3Na+/2K+-ATPase or Renal H+-ATPase (Can push up X1000) • 2) V-type ATPase: Common pump on organelles • Not directly phosphorylated, just use energy released from ATP • Can push substrates up gradients of X10 to x10,000! • 3) F-type ATPase: Common pump on bacteria, chloroplasts and mitochondria! (primitive) • Has H+ and ATPsynthase parts! • Use to GENERATE ATP! • 4) ABC-type ATPase: Common to eukaryote PM • Help move large molecules like drugs, sugars, aminoacids! • Very important in medicine!

20. Sometimes several types of active transport work at the same time, occasionally in opposition to each other! Evolution has created some mixed up ways to get work done in cells given the tools at their disposal! F-type ATPase

21. Secondary Active Transport: We often link transport of large molecules to gradients established by the Na+/K+-ATPase in the gut and kidney (below)!How does GatorAide help in this regard in the gut?

22. By establishing a Na+ gradient with Na/K-ATPase, we create an anti-port system to move H+ out of body during acidosis!Protons (H+) can also be directly pumped by a different kind of ATPase in the collecting duct to acidify your urine!

23. The 3Na+/2K+ ATPase pump is the single most important enzyme in all cells with regards to maintaining membrane concentration gradients! How does the pump work?

24. Delta G and the cost of Concentration Gradient Maintenance! • Na+ Outside: 140 mM Inside: 4 mM • K+ Outside: 3 mM Inside: 150 mM • Pump Gradients seldom exceed 50:1 • Energy per mole pumped is about 2 kcal/mol • Energy from ATP hydrolysis: -7.3 kcal/mol ATP Hydrolysis makes process thermodynamically feasible!

25. Delta G values and calculations also apply to concentration gradients across a membrane.

26. 10 Repeats Assignment for Wednesday Oct 3rd: 10 pts • Glycolysis and Gluconeogenesis: Write using pen or pencil the names of all enzymes, reactants, and products required for one glucose to be oxidized to two molecules of pyruvate in the cytosol (glycolysis). Show the additional enzymes required for two pyruvates to be converted back into a single glucose (gluconeogenesis). Show the enzymes used to create two lactic acid molecules in cells lacking mitochondria or in cells that are hypoxic. Write this same thing out (by hand) and repeat this nine more times (10 times total). Friday 20 pt Quiz covering CH 7 and CH 8: