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Cell membrane history (prob not relevant)
1) Schleidew + Schwann
- Coined the term barrier
2) Overton
- Stained cells with dye (hydrophilic or hydrophobic)
Determined that lipid dyes stain cells concluding that the barrier is a lipid in nature.
3) Bernstein
- Hypothesized that cells have an electrolyte interior (negative inside), which was true
4) Langmuir ***
- Put phospholipids into a trough
- Moved a molecule through the phospholipids
- Measured the resultant pressure
Determined that phospholipid layer is mono or bi
5) Gorter + Grendel
Used RBCs
- Extract all phospholipids
- Measure surface area
Messed up SA calculation and said membrane is only bilayer
6) Mudd + Mudd
- RBCs prefer oil mixture
- WBCs prefer water
Determined that there could be proteins in blood cells
7) JD Robertson
- Used TEM and thought he saw lipid bilayer
This was actually OsO4, a heavy metal
TEM can’t be used to see lipid bilayers
Did determine that outer layer is glycosylating (glyco-protein)
4 critical experiments
1) Freeze Fracture (Dan Branton)
- FF on RBCs, found weird bumps
- Did FF on liposomes (pure, no proteins, mono/bilayer), had no bumps
Determined that mosaic of bumps are proteins
Determined that the P face (protoplasmic, inside) had more bumps than the E face (ectoplasmic, outside)
2) Cell Fusion
Requires: 2 cells, 2 mAbs, 2 fluorochromes
*Byproduct of trying to fuse b-cell with a myeloma cell, creating a hybridoma cell (mAb)
- Mouse cell + human cell fuse with membrane proteins creating a heterokaryote (2 different nuclei)
- Cell goes from being half red/green to fully yellow
Suggests that cell membrane proteins are fluid within plane of membrane
3) Cell patching/capping
Tackles experiment 2, but uses 1 mAb, 1 fluorochrome, 1 cell.
- Uses bivalent mAb ‘in situ’ to cause immunoprecipitation in membrane after binding to 2 fab regions
Suggests that proteins are fluid within the membrane
4) FRAP
- Uses con A to bind to carbohydrate groups, binds to all cell membrane proteins for labelling
- Graph produced shows that 50% of proteins are mobile, rest stays in place
This happens because of ECM or stuck in place by cytoskeleton
5) Fluid-mosaic model - Used to represent 3-D biological membrane
RBC Utility
1 - Plentiful and easy to obtain
2 - Can be separated easily (centrifuge)
3 - No contaminating organelles inside cell
4 - Pure population of cell membranes can be formed
5 - Few proteins in cell membrane
6 - RBC Ghosts made (hypertonic lysis removes hemoglobin), these are then disrupted inside out/outside in.
Can then test what proteins are accessible
RBC membrane model
1) Alpha + beta spectrin
Major component of RBC cytoskeleton, maintain biconcave shape
Phospholipid Categories
Measures solubility by radioactively tagging all types to see if it can pass membrane
1) phosphoglycerides (ex. phosphatidyl sereine-annexin 5)
Inner leaflet flips into outer leaflet when cells are going through apoptosis, stained with annexin 5
2) Sphingolipids (ex sphingomyelin [common in nerve cells, because it increases electrical resistance, important for resting/action potentials])
Found in excitable cells (ex. neuron, cardiomyocytes) that can change their membrane potential
3) Sterols (ex. cholesterol)
Important in maintaining membrane fluidity, cholesterol is mostly stored in membranes
Statins are used to lower LDL level, inhibits HMDA synthesis which produces cholesterol
Phospholipid Synthesis
Phospholipids made in ER
Flippase flips phospholipids (ABcB4 or ATPase)
- is put into a bilayer liposome
- Quencher is asked to fluoresce (can’t get into vesicle) outside membrane leaflet
- ATP introduced, now quencher can enter cell due to leaflet flipping reducing fluorescence
Phosphatidyl ethanolamine, found in curves of membrane
Cell membrane proteins
1) Integral membrane proteins - not easily washed off, proteins that go through the membrane
2) Peripheral membrane proteins - easily washed off
3) Lipid/phospholipid anchor proteins
Farnesyl + Myristic anchors
- Cancer can potentially be inhibited by blocking access to these anchors saving the respective cell from the oncogenes
Glycophorin
- glycosylation, outside portion (remember con-a)
- amino acids, inside the membrane are all hydrophobic (stay inside due to non covalent bonds)
Channels + Pumps
1) ATP powered pump
Uses 70% of ATP (ex. nerve or kidney cells) to pump against the gradient
2) Channels
No ATP used, fastest, passes ions down gradient
(a) Ligand-gated (ex. nicotinic acetylcholine channel)
Process: Ligand is ACH, Na+ in and K+ out
(b) Voltage gated calcium channel in neurons
Process: action potential (+55mV) opens the channel in neurons causing a flood of calcium, this triggers vesicle fusion
