Questions regarding Biomembranes or Antibodies????????
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Transcript of 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.