Two Approcahes To Studying Cell Membranes

1) Microscopy
a. Electron Microscopy
b. Light Microscopy
i. Fluorescence Microscopy
2) Membrane Fractionation
In Electron Microscopy you take a piece of cell/tissue and use a
chemical to crosslink the molecules in the cell/tissue (to avoid their
floating around). Remove water by dehydrative steps, and then embed
them in a plastic resin (to make a plastic block). This preserves the
molecular structure of the membrane (despite the specimen being
dead). Subject it to very thin slices into the cell (with diamond cutting
tool) at around 100 nm thickness. Then you stain the specimen on a
ribbon (that attach to parts of the cell) and put it into an electron
microscope. The electron beam is fired onto the specimen that the
stain scatters and you can observe the reflection of the scattered
electrons.
Light Microscopy is where there have been recent advances, and is
where the future of microscopy may lie. Fluorescent microscopy
involves a green pigment isolated from a jellyfish (Green Fluorescent
Pigment - GFP). You isolate the gene from GFP and use it to append it
to any other protein that would carry the GFP molecule with it allowing
the observation of where it is in the cell. Its like you tag a protein
visually and can watch it in real time. (On a side note the person who
found the technique is now a truck driver and people who picked up his
work won a Nobel Prize). Of course, fusing GFP with a protein can
cause it to behave differently.

In Membrane Fractionation you take tissue and subject the tissue to a

technique that ruptures the cell (usually intense homogenization). The
process involves putting the tissue in a tube and then sticking a tightly

fitting (usually Teflon) pestle inside and back and forth. This gives you
a lysate containing many membrane fragments (e.g. plasma
membrane, endoplasmic reticulum, nuclear membrane, mitochondrion
membrane, lysosome membrane, golgi apparatusetc). These are
floating around in organelles as a soup. You then can gently use a
centrifuge to spin it lightly, the first thing to fall out of suspension is
the nuclear membrane. On the other hand you need a higher level of
RPM to settle bubbles of membranes known as vesicles. You can of
course use differential centrifugation (spinning at various speeds).
Membranes largely consist of lipids, making it light. But pieces of the
ER contain ribosomes in the membrane (for secretory protein
production) have higher buoyant density. You can use a sucrose
gradient to help you isolate the high buoyant density particles from
the low buoyant density ones. To do this you just create a gradient with
the higher concentration of sucrose at the bottom of the tube and then
pour the lysate soup at the top and put it in a centrifuge until it
reaches equilibrium. This helps as the higher buoyant density particles
will be at the bottom of the tube. The two processes of sucrose
gradient and differential centrifugation can leave you with the ability to
separate two quite homogenous types of membranes.

Properties of Membranes
Membranes are Selectively Permeable
They expel charged particles/polar particles/expel sodium ions
etc. Hundreds or thousands of permeases or transport channels
allow the cell to perform these tasks. Membranes alone are generally
impermeable to ionic/charged particles.
Also theres oxidative phosphorylation in the mitochondrial
envelope, in fact its possible that mitochondria evolved from some
sort of symbiosis of eukaryotic cells and primitive bacterial cells. The
outer membrane has stable channels known as porins that are
essentially just pores, theyre selective only by size (~100 daltons) and

are a method of passive transport. The inner membrane is very active

in the transport of charged particles (the stuff inside the inner
membrane is known as the matrix). When theres an excess of
proteins on the outside of the mitochondrial matrix, they drive the ATP
synthase (a protein on the surface of the mitochondrial envelope) to
produce ATP. This would be intrinsically impossible if the surface of
the membrane was permeable naturally.

Membrane are Mechanically Deformable

The can divide, they can send out vesiclesetc They can do this
because they arent rigid, but are deformable. They are because
they are largely composed of lipids.
They also serve in cell-cell communication.
Messages are conveyed by a secretory method, and go through a
distance and combine with the distal cell. This is known as signal
transduction.
The simplest membrane to study, is the red blood cell
membrane. You can isolate the red blood cells with centrifugation of
blood, and then since there are no intracellular organelles, you have no
additional membrane to worry over. When red blood cells are
submerged in distilled water, the difference in osmotic pressure
between the hemoglobin cytoplasm and the distilled water is sufficient
to cause the red blood cell to lyse (kind of explode). You are left with
the ghost of the red cell. In some situations the shape is retained. If

you centrifuge again, you get a pellet that has no soluble proteins
(hemoglobin is left in another fraction). If you suspend the pellet in a
mixture of chloroform and methanol, you can extract the lipids that are
in the membrane.
Red Cells Lyse -> Membranes + Chloroform + Methanol
Mixture is set s.t lipids and cholesterol are soluble, and proteins are
denatured (the polypeptide unfolds). You get a milky white, kind of like
what you get from scrambling eggs.
The main component of membranes in cells are phospholipids
(especially some specific animal cells)
As you can see, the
phospholipid is built on a
base of glycerol.
Youll notice that there are
two carbons that are
esterified (meaning the
hydrogen of an acid is
replaced my an alkyl or
other organic group) to
chains of hydrocarbons
known as fatty acids.
These are very non-polar
Carbon three, is esterified to
a phosphate compound
which is then connected to
a head group which may
vary (here its composed of
CH2CH2NH3) but is usually very polar.

Fatty acid kinds:

C16 is common, found in all cells. Its known as palmitate (Palmitic
Acid) and tends to wiggle around, depending on temperature.
Sterate (Steric Acid) is also saturated, but its slightly larged at C18.
Oleate (Oleic Acid) is also common, and may have a kink rendering it
less flexible because of a double bond leading to an unsaturated fatty
acid. Its also C18

An amino alcohol known as sphingosine, its uniquely eukaryotic and

bears an unsaturated hydrocarbon chain. The amino group can be
modified by another fatty acid, to create a molecule containing two
fatty acids (like a phospholipid)
If you look at the mobility of the phospholipid chains, you can see
that the mobility is greatest at the end of the chain (so center of the
bilayer). An experiment thats useful in studying membranes relies on
the observation that if you take a lipid from a red cell after the
chloroform methanol method (so you have the lipids) and you have
them dried on the bottom of a tube as a film, if you insert buffer (via
pipette) and shake the tube vigorously, the lipids wont flow to the
air/water interface but will form spontaneously circular structures
known as vesicles or liposomes, which are just continuous bilayers.
So you can make a replica membrane just by adding buffer and
shaking. You may even get Russian doll-like membranes
(membraneception), or if you shake vigorously you get smaller
vesicles. But you have to see if they are impermeable.
Now we need an experiment to test the
permeability/impermeability of the membrane. Just do what was
previously mentioned, but include an tracer isotope of sodium
(unstable + radioactive), which is a cation that usually require
transport proteins to pass in and out of the membrane. You can be sure
that at least some of the sodium cations are trapped within the
membrane vesicles. Then if you centrifuge the sample so that the
vesicles pellet at the bottom of the tube, and some sodium is left
above it. If you look at the concentration of the radioactivity it should
be the same at both the top and in pellet. (If you add calcium / proteins
the vesicles may fuse. But it wont change the experiment. The sole
worry is that they may break). Then you replace the supernatant
buffer with a new buffer. You can see that the sodium is always
contained in vesicles, despite the surrounding buffer being absent of
the cation.
A property of fatty acids is that their viscosity decreases when
temperature increases (as it enters a liquid state). But the more double
bonds (kinks) a fatty acid has, the less temperature (TM) is required for
it to go to a liquid state.
Interestingly, in some bacteria there are enzymes that can adjust
to the ambient temperature by adding or removing double bonds in the
fatty acids, therefore they remain able to function despite the
temperature.

Peripheral Membrane Proteins adhere (usually) to the

cytoplasmic side of the membrane. They can be removed by changing
the quantity of salt, pH, or the quantity of divalent cations.
Spectrin is a protein that forms a continuous network of a red
blood cell giving the red blood cell its distinctive state. Mutations of it
can cause red blood cell shape diseases, such as sickle cell or
hereditary spherocytosis.
Hereditary Spherocytosis is a disease of the red blood cell shape.
They, as the name implies, become round. These have great difficulty
penetrating capillaries. They
Integral proteins are imbedded into the bilayer. So at least part of
the chain is in physical + chemical contact with the bilayer. They cant
be removed unless you use something like a detergent to solubilize
the membrane and separate the lipids from the proteins. You shouldnt
use a detergent that denatures everything. (SDS or Sodium dodecyl
sulfate that is in many cleaning/hygiene products does just that.
Denatures everything.) To isolate a membrane protein you want use a
detergent that removes the phospholipids that form an outer shell
without invading the interior of the membrane. SDS will go into the
interior of the protein and cause it to unfold. Non ionic (milder)
detergents will bind to the surface and will replace certain chains of the
proteins with a detergent molecule (e.g. Triton X-100).
When you add a triton to a buffer, a micelle forms. It has an
apolar chain in the middle of it, while the head groups are polar. These
micelles go into the membrane, and kind of surround the membrane
protein. The membrane protein will then be lifted right out of the
membrane. You can hopefully keep the protein purified and active to
study it. You have to keep detergent though, if you remove detergent
from whatever the protein is in though, the proteins hydrophobic part
will force it to crash out of solution.

Lets suppose weve purified ATPase in the mitochondria. The

best way to study it here is to put it back into the membrane. Turns out
you can remove the detergent through the experiment with the
vesicles that proves membranes are impermeable, of course to keep
the protein active some must remain. If you take the detergent
complex and agitate it the protein pops into the vesicle, and now you
have an enzyme containing membrane. But the ATP binding site may
(depending on luck) then be on the inside (or outside) of the vesicle
during the constitution of a membrane vesicle.
This leads us to try to determine the fraction of ATPase binding
sites on the inside of the vesicle or the outside of the vesicle. E.g. 50
are in and 50 are out, the method of determining this is by adding
radioactive ATP (phosphate is labeled with an isotope ex. (P32)). Then
you have to do a latency experiment. So find the rate at which ATP is
hydrolyzed in the absence of detergent, and with detergent. Divide the
two rates to get the fraction of molecules exposed. If 60% are evident,
then 40% are latent (cant be accessed).
There are sugars (e.g. glycolipids) and other carbohydrates,
usually branch chains on proteins. The bonds between the sugar and
the protein are called glycosidic? bonds.
Plant proteins called lectin, they are primitive antibodies found in
storage vacuoles. Bacteria, and other fungal pathogens have sugar
residue on their membrane surface. Lectins stick to the sugar residues
and cause them to cluster and clump, which reduces their ability to
infect the plant.
Con-A is a plant lectin with a preference to binding glucose and
nanose. Nanose is found on the outer surface of red blood cells, so if
you were to bind a red cell with Con-A, the nanose on the surface
would be bound. In fact through attaching gold to the lectin, if you
were to allow lectin to be on both the inner and outer surfaces of the
membrane, you would see it only binds to anything on the outer
surface, because thats where the nanose is.
Enzymes like trypsin can remove the proteins on the surface of
some cells, this could help determine if the membrane protein has all
or some of its amino acid chain exposed to the surface.
Now suppose we have five membrane proteins:
1) Peripheral Membrane Protein, facing cytoplasm. Attached by
electrostatic interactions.
2) A portion of its structure is embedded in the external leaflet in the
bilayer and is exposed to the outer surface.
3) Embedded in the bilayer. Its also exposed to both the outside of the
cell as well as to the cytoplasm.

4) Same as protein 2, except its exposed partially to the cytoplasm

instead of the outside of the membrane.
5) Its bound peripherally via an interaction with protein 4.
Now theres going to be a simple experiment. Expose intact cells to
trypsin (it says protease on the board also he says it once but calls it
trypsin every time after; trypsin makes more sense here since it frees
proteins) with/without detergent. Centrifuge, and leave any shaved
away peptides (found in the supernatant). Take the pellet and run an
SDS gel.