Questions regarding Biomembranes or Antibodies????????

FRAP Cell fusion Liposomes Immunoprecipitation SDS PAGE GFP fusions Western Blots Polyclonal Ab Protein fractionation Micelles Detergent extractions Monoclonal Ab

Intracellular 5-15 millimolar sodium’ 140 millimolar potassium Less than micromolar calcium Extracellular 145 millimolar sodium 5 millimolar potassium 1 millimolar calcium Cells are electrically neutral although the charges are not evenly distributed.

The gradients are established by transporters and pumps. Selectivity

Bilayers (not cell membranes) are a billion times Memorize this. Bilayers (not cell membranes) are a billion times more permeable to water than to sodium

(Facilitated diffusion) Solutes cross membranes by either passive or active transport. Here’s how: (Facilitated diffusion) You already read about bacteriorhodopsin,

Concentration and electrical gradients drive the movement/direction. e.g. Glucose transport-bidirectional, electrical is irrelevant Add the sarcoplasmic reticulum- Calcium pump

An example of active transport: Calcium pump on the sarcoplasmic reticulum

Passive: A Uniport, Glucose Carrier

Km D-glucose 1.5 mM; L-glucose 3000 mM

GLUT1 High to Low (RBC) 12 a helices 2% of RBC protein Specific for D glucose Pay attention to these arrow heads 12 glucose binding triggers conformational change. 23 glucose now facing the cytoplasm 3—4 glucose can be released to cytoplasm 45 glucose dissociation triggers return to original conformation How can this continue to run?? Answ: hexokinase

Good example: potassium

Three common ways to couple energy release with transport:

P type Calcium ATPases sodium-potassium pump V-type ATP production- more on this later ABC Transporters lots in bacteria MDR proteins in selected cancer cells malaria- chloroquine pump CF yeast mating factor class I MHC immune response

Pumps move solutes against their electrochemical gradient- obviously they require energy

across the plasma membrane. Operates constantly. A major use of ATP in cells is setting the sodium and potassium gradients across the plasma membrane. Operates constantly. Large electrochemical Balanced electrochemical

10 milliseconds!!

[Na+]out ~400 mM [K+]out ~ 4-20 mM [Na+]in ~12-50 mM [K+]in ~400 mM E1’ E1 E2 [Na+]in ~12-50 mM [K+]in ~400 mM E2 Km Na+ ~ 0.6 mM Km K+ ~ 0.2 mM

Na+/K+ ATPase maintains the intracellular Na+ and K+ in cells Evidence that this pump is responsible for coupled K+/Na+ movement: Ouabain blocks the ATPase and Na+/K+ movement Liposome reconstitution demonstrated Na+/K+ exchange The mechanism is similar to the Ca++ ATPase but not exactly the same Coupled transport and phosphate hydrolysis drives “K+ in” conformation

Coupled transporters can use the energy stored in gradients.

Polarized epithelial cell

two

AE1 protein, a Cl-/HCO3- antiporter is crucial to CO2 transport in RBC i.e an anion transporter. No net charge movement. Concentration only. Movement of CO2 from peripheral tissues (systemic capillaries) to lungs. Carbonic anhydrase in blood converts CO2 to water soluble bicarbonate/ . i.e CO2 is loaded into cells and carbonic acid is pumped out. Release of CO2 in the lungs because O2 drives carbonic anhydrase in reverse. CO2 in the lungs moves into RBC with Cl- exchange.