Two membrane preparations have been derived from the plasma membrane of erythroc
ID: 48537 • Letter: T
Question
Two membrane preparations have been derived from the plasma membrane of erythrocytes and are present in a buffer solution: (1) right-side-out membrane vesicles, in which the exoplasmic leaflet of the vesicle faces the buffer (equivalent in orientation to that of the exoplasmic leaflet of the intact plasma membrane); and (2) inside- out membrane vesicles in which the exoplasmic leaflet faces the interior of the vesicles.
A- [5pts] Annexin V is a protein that binds to phosphatidylserine, one of the phospholipids present in cell membranes. Fluorescently labeled annexin V is mixed with each vesicle preparation. The vesicles are washed to remove unbound annexin V and then examined by fluorescence microscopy. Presence of annexin V is detected by green fluorescence, as shown above. What can be deduced from these data?
B- [3pts] A monoclonal antibody has been prepared against band 3, an integral membrane protein of the erythrocyte plasma membrane. The antibody recognizes an epitope on the carboxy terminus of band 3. Incubation of the two membrane vesicle preparations with this antibody following by staining with a fluorescently labeled secondary antibody generates data similar to that shown in the micrographs above. What can you deduce about the structure of band 3 from these data?
The two vesicle fractions are either untreated (–) or are incubated with the protease trypsin (+). The membrane vesicles are then washed, the membranes solubilized and the proteins denatured and analyzed by SDS gel electrophoresis and Western blotting using polyclonal antibodies against glycophorin A, an erythrocyte plasma membrane protein. The following data are obtained:
C. [5pts] What can be deduced from these data about the structure of glycophorin A? Explain
Right-side-out vesicles ROVs) Inside-out vesicles (IOVs)Explanation / Answer
A- right side out vesicles are impermeable to small molecules.annexin V binds to phosphatydyl serin. therefore annexin V cannot cross the right side out vesicle. so it will ultimately bind the phosphatydyl serine, which is present in the leaflet facing to the buffer.hence, annexin V staining will be more prominent in the right due to higher binding of the annexin V. therefore phosphatydyl serine is more found in cytoplasmic leaflet.
B- binding of theannexin V was found to the inside of the membrane vesicle.
glycoporin A is mostly a transmembrane protein, therefore the domains are accessible to cytoplasmic and exoplasmic sides.the exoplasmic domain of glycoporin A could be larger than the cytoplasmic domain. because the MW of the part of the protein which is resistant to protease is larger when the inside out vesicvles are digested.
trypsin also cannot cross the bilayer membrane
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