questions regarding biomembranes or antibodies????????

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

Immunoprecipitation SDS PAGE

Western BlotsPolyclonal Ab

Monoclonal Ab

GFP fusions

Protein fractionation

Detergent extractions

Liposomes

Micelles

Cell fusionFRAP

Intracellular5-15 millimolar sodium’140 millimolar potassiumLess than micromolar calcium

Extracellular145 millimolar sodium5 millimolar potassium1 millimolar calcium

Cells are electrically neutral although the charges are not evenly distributed.

The gradients are established by transporters and pumps.

Selectivity

Memorize this.

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

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 reticulumAn 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

Pay attention to these arrow heads

12 glucose binding triggers conformational change.23 glucose now facing the cytoplasm3—4 glucose can be released to cytoplasm45 glucose dissociation triggers return to original conformation

How can this continue to run?? Answ: hexokinase

GLUT1 High to Low (RBC)12 helices2% of RBC proteinSpecific for D glucose

Good example: potassium

Three common ways to couple energy release with transport:

P typeCalcium ATPasessodium-potassium pump

V-typeATP production- more on this later

ABC Transporterslots in bacteriaMDR proteins in selected cancer cellsmalaria- chloroquine pumpCFyeast mating factorclass I MHC immune response

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

A major use of ATP in cells is setting the sodium and potassium gradientsacross 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

Km Na+ ~ 0.6 mM

Km K+ ~ 0.2 mM

E1

E1’

E2

E2

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+ movementLiposome reconstitution demonstrated Na+/K+ exchange

The mechanism is similar to the Ca++ ATPase but not exactly the sameCoupled 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.