Lab 5: MEMBRANE FUNCTION: MEMBRANE TRANSPORT MECHANISMS
(PLEASE “DO NOT” USE THE MICROPIPETTES FOR THIS LAB)
An important and intensively investigated biological phenomenon is the process of cellular
transport. This process allows the composition of the cytoplasm to be regulated and to be
adapted to whatever conditions exist in the internal and external environments. Our current
model for explaining this process is that membranes are selectively permeable. The model
suggests the membrane structure is a phospholipid bilayer in which a number of proteins and
other organic molecules are embedded. This structure allows the membrane to act as a “fluid
mosaic” interface on which mechanisms of selective transport act to regulate what goes into and
out of the cell.
This unique membrane structure helps explain its selective permeability. Materials that cross the
cell membrane must either squeeze between individual phospholipid (e.g. pass through pores in
the phospholipid bilayer), dissolve in the phospholipid membrane, or complex with specific
mobile molecules embedded in the membrane that act as carriers to shuttle materials in or out of
the cell. Therefore any molecule that can enter or leave a cell must have one or more of these
characteristics:
a. Be small enough to pass through the membrane’s pores
b. Have no charges or polarities that are repulsed by the molecules around the pores.
c. Be lipid soluble.
d. Be able to complex with a carrier molecule.
The work of moving ions and molecules uses energy from one of several sources—kinetic
energy of molecular motion (heat) or the potential energy of an electrochemical gradient; or
metabolic energy acquired by breaking down ATP. When molecular motion is the source, the
transport is called passive, and when ATP is the source, it is called active.
A number of transport mechanisms are found to occur in living systems. These include bulk
flow, diffusion, osmosis, facilitated diffusion, primary active transport, secondary active
transport, and active transport using vesicles (e.g. endocytosis, receptor-mediated endocytosis,
and exocytosis).
Osmosis is the special case of diffusion where the particles moving are water molecules and the
movement is across a semi-permeable membrane. The diffusion of water will always occur from
an area of higher water concentration to an area of lower water concentration. However, the
relationship between the two solutions on either side of the membrane is usually defined on the
basis of their solute concentration differences (e.g. differences in molarity, or differences in %
concentration, etc.). Of the two solutions, the one with the fewer number of solute molecules is
said to be hypotonic to the one with the greater number of solute molecules. Conversely, the
solution with the greater number of solute molecules is said to be hypertonic to the one with the
fewer number of solute molecules. If the two solutions have an equal number of solute
molecules, they are said to be isotonic to one another. These tonicity relationships are only used
when comparing one solution to another.
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A. Osmosis
In this exercise you will determine the direction and rate of osmosis. A dialysis tubing of 25angstrom pore size will be used that is selectively permeable to water but not to molecules the
size of sucrose.
Procedure:
1.
Fill three beakers with tap water.
2.
Obtain three pieces of soaked dialysis tubing. For each piece of tubing, twist one
end and fold the end over. Tie it tightly with string.
3.
Add 5 ml of tap water to one of the tubes. Twist the other end, fold it over, and
tie it tightly with string. Place it on a paper towel in front of the beaker labeled
“water.”
4.
Following the same procedure in step 3 above, add 5 ml of a 10% sucrose solution
to a second tube and 5 ml of a 60% sucrose solution to the third tube. Again,
make sure that you fold over and tie the ends of the tubing tightly to prevent
leakage. It is OK to have air spaces inside the sac. (Briefly submerge the bags in water
and then properly dry them before taking your first weight measurement. Why is this important?)
5.
Gently dry each bag (use paper towels). Check for leaks by blotting against dry
paper towels. Record the weight of each bag in the data table provided.
6.
Place each bag into a beaker of water. Make sure each is submerged. After 10
minutes, remove the sacs from the water, dry the surface (carefully, but
thoroughly), and weigh them. Record the weights.
7.
Repeat step 6 at ten minute intervals until no further change in weight is detected
OR 60 minutes (which ever occurs first). However, this should take about one
hour.
8.
Graph the data collected. Plot time on the x axis and weight on the y axis. One
graph containing all three plots should be prepared; simply use different symbols
for the data points of each sac, or use different colored lines, etc. Provide a
legend to identify each plot by the code used. Lastly, be sure to properly title the
graph and label both axes (with units and date of experiment).
9.
Determine the rate of osmosis for each condition. To do this, you must determine
the change in weight per unit of time for the period of active osmosis. If
equilibrium was reached for all three conditions, the change in weight for the time
required to achieve it can be calculated.
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Title: _____________________________________________________ Date: ___________
Bag Weight (g)
sac
content
0
min
10
min
20
min
30
min
40
min
50
min
60
min
Osmosis Rate*
(g/min)
water
10%
sucrose
60%
sucrose
* The Osmosis Rate can be determined by taking the weight of the bag of solution at 60
minutes minus the weight of the bag of solution at 0 min and dividing the difference by 60.
B. Transport in a Living System
In this lab exercise you will perform a simple procedure to explore the transport of
neutral red and sodium carbonate by yeast cells. Other than procedural details, no further
information is given. Based solely on your observations, your understanding of the various
transport mechanisms, and applied reasoning, you are asked to propose a mechanism of transport
for these two molecules.
Procedure:
1. Label three 125 ml Erlenmeyer flasks A, B, and C. Place 10 ml of 0.75% sodium
carbonate into each flask. Record the color of the sodium carbonate solution.
2. Add 10 ml of neutral red to each flask. Record the color of neutral red and the
mixture of sodium carbonate and neutral red.
3. Add 5 ml of yeast suspension to flasks A and B. Mix (by swirling) the contents of
these flasks for 2-3 minutes. Record the color of the solution.
4. Filter 5 ml of the contents of flask A into a clean test tube using a small funnel and
filter paper. Swirl flask just prior to sampling to ensure obtaining a uniform sample.
Record the color of the filtrate and material remaining on the filter paper. Slowly add
2-3 ml of neutral red (this should be approximately the same volume as the filtrate)
directly to the filtrate obtained above (do not add through the filter paper). Record
any color change.
6. Place flask A on a hot plate and heat the yeast suspension for 2 minutes or until the
color changes. Record the color of the solution.
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7. Place 5 ml of yeast suspension in a polypropylene centrifuge tube (no cap). Fill a
beaker with ~100 ml of tap water and cover the beaker with a piece of foil. Place the
beaker on a hot plate and wait until boiling. Place the polypropylene tube into the
boiling water and boil the yeast suspension for 5 minutes. Add the boiled yeast
solution to flask C and mix for 2-3 minutes. Record the color of solution. Compare
with that of flask B. Filter 5 ml of the contents of flask C. Record the color of the
filtrate and material remaining on the filter paper.
Title: _____________________________________________________ Date: ___________
Flask
Na2CO3
(SC)
Neutral
Red
(NR)
NR + SC
NR/SC +
Yeast
Filtrate
Material
on
Filter
Paper
Filtrate +
NR
Flask
after
boiling
A
B
C

Limit your color choices to: RED, YELLOW/GOLD, or COLORLESS.
After drawing your graph, write a hypothesis to explain the data in Part 1 (dialysis bags) and Part
II (yeast). Propose any alternative tests that would confirm your hypothesis.
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