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MEMBRANE CONFERENCE

PROBLEM #1
The primary role of platelets is to control blood
clotting. When they encounter the exposed
basement membrane (collagen fibers) of a
damaged blood vessel or a newly forming fibrin
clot, they change their shape from round to spiky
and stick to the damaged area. At the same time
they begin to secrete serotonin and ATP, which
accelerate similar changes in newly arriving
platelets, leading to the rapid formation of a clot.
The platelet response is regulated by protein
phosphorylation. Significantly, platelets contain
high levels of two protein kinases: C-kinase
(protein kinase C), which initiates serotonin
release, and myosin light-chain kinase, which
mediates the change in shape.
When platelets are stimulated with thrombin,
the light chain of myosin and an unknown protein
of 40,000 daltons are phosphorylated. When
platelets are treated with a calcium ionophore,
only the myosin light chain is phosphorylated;
when they are treated with diacylglycerol, only the
40 kd protein is phosphorylated. Experiments
using combinations of calcium ionophore and
diacylglycerol show that the extent of
phosphorylation of the 40 kd protein depends only
on the concentration of diacylglycerol (Figure 1510A); however, serotonin release depends on
diacylglycerol and the calcium ionophore (Figure
15-10B).
A. Based on these experimental observations,
describe the normal sequence of molecular
events that leads to phosphorylation of the
myosin light chain and the 40 kd protein.
Indicate how the calcium ionophore and
diacylglycerol treatments interact with the
normal sequence of events.
B. Why do you think serotonin release requires both calcium ionophore and
diacylglycerol?

PROBLEM #2
After prolonged exposure to hormones that bind to -adrenergic receptors, cells
become refractory and cease responding. To examine this desensitization
phenomenon, you use a newly developed reagent, CGP-12177, which is a hydrophilic
molecule that specifically binds to -adrenergic receptors. In contrast to the binding of
dihydroalprenolol, which is hydrophobic, CGP-12177 binding exactly parallels the
decrease in hormone-dependent adenylyl cyclase activity observed in extracts from
cells treated with isoproterenol for increasing times (Figure 15-16). To understand the
difference in receptor binding by these two molecules, you lyse untreated cells and
isoproterenol-treated (desensitized) cells, fractionate them by centrifugation through
sucrose-density gradients and measure binding by dihydroalprenolol and CGP-12177
(Figure 15-17). In addition to ligand binding, you also measure 5'-nucleotidase activity,
which is a marker enzyme for the plasma membrane.
A. Give an explanation for the differences in binding
by dihydroalprenolol and CGP-12177.
B. What do you think might be the basis for
isoproterenol-induced desensitization in these
cells?

PROBLEM #3
Insulin is a small protein hormone that binds to a receptor in the plasma membrane
of fat cells. This binding dramatically increases the rate of uptake of glucose into the
cells. The increase occurs within minutes and is not blocked by inhibitors of protein
synthesis or glycosylation. Therefore, insulin must increase the activity of the glucose
transporter in the plasma membrane without increasing the total number of transporters
in the cell.
The two experiments described below suggest a possible mechanism for the insulin
effect. In the first experiment, the initial rate of glucose uptake in control and insulintreated cells was measured, with the results shown in Figure 1. In the second
experiment, the concentration of glucose transporter in fractionated membranes from
control and insulin cells was measured, using the binding of radioactive cytochalasin B
as the assay as shown in Table 1.
A. Deduce the mechanism by which glucose
transport is increased in insulin-treated
cells. (Hint: cytochalasin B binds specifically
to glucose transporters).
B. Transport proteins, like enzymes, can be
characterized by the kinetic parameters Km
(the concentration of substrate at which the
rate of transport is half-maximal) and Vmax
(the rate of transport achieved at saturating
substrate concentration). Does insulin
stimulation alter either of these kinetic
properties of the glucose transporter? How
can you tell from these data?

Table 11-2 Amount of Glucose Transporter Associated with the Plasma Membrane and
Internal Membranes in the Presence and Absence of Insulin (Problem 3)
Bound 3H-Cytochalasin B
(Cpm/mg vesicle protein)
_______________________________
Untreated cells
Treated cells
Membrane Fraction
(- insulin)
(+ insulin)
Plasma membrane
880
4480
Internal membranes
4070
480

PROBLEM #4
Much of our knowledge of the topography of plasma membrane components has
been gained by study of the red blood cell. Since these cells contain no intracellular
organelles, the plasma membrane can be easily isolated free from intracellular
components by lysis in a hypotonic solution. The membranous sacs or ghosts so
obtained can be sealed under certain experimental conditions (sealed right-side-out
ghosts). They can also be turned inside out and sealed, thereby making the original
cytosolic side accessible from the outside.
Enzymatic digestion of sealed right-side-out red
cell ghosts was originally used to determine the
sidedness of the major membrane-associated
proteins: spectrin, band 3, and glycophorin. These
experiments made use of sialidase, which removes
sialic acid residues from protein, and pronase, which
cleaves peptide bonds. The proteins from normal
ghosts and enzyme-treated ghosts were separated
from SDS polyacrylamide-gel electrophoresis and
then stained for protein and carbohydrate (Figure 105).
A. How does the information in Figure 10-5 allow
you to decide whether the carbohydrate of
glycophorin is on the cytoplasmic or external
surface, and how does it allow you to decide
which of the red cell proteins are exposed on the
external side of the cell?
B. When you show your deductions to a colleague,
she challenges your conclusion that some
proteins are not exposed on the external surface
and suggests instead that these proteins may be
resistant to pronase digestion. What control
experiment can you propose to test this
possibility?
C. How would you modify this enzymatic approach
in order to determine which red cell proteins span
the plasma membrane?

PROBLEM #5
You are studying the binding of proteins to the cytoplasmic face of cultured
neuroblastoma cells and have found a method that gives a good yield of inside-out
vesicles from the plasma membrane. Unfortunately, your preparations of inside-out
vesicles are contaminated with variable amounts of right-side-out vesicles. Nothing you
have tried avoids this variable contamination. A friend suggests that you pass your
vesicles over an affinity column made of lectin coupled to solid beads. What is the
point of your friends suggestion?

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