Question

3b and 4 please

Exercise 9-55 3. You wish to determine the distribution of phospholipids in the plasma membrane. You use right-side out and inside out red blood cells. You treat the cells with SITS, a fluorescent dye that reacts with primary amino groups and two different phospholipases. The results are sum- marized below (+reacted did not react). Sphingolipase Sea snake venom SITS Right side Inside out Right side Inside out Right side Inside out out out PPhosphatidycholine P Phosphatidyl-ethanolamine SSphingomyelin P9Phosphatidylamine From these results deduce the distribution of the four principal phospholipids in membranes. Is the cytosolic face of the plasma membrane the same as the exoplasmic face? a. b. Why did you use red blood cells for this experiment? Assume that you lysed red blood cells and let the membranes reform knowing that some will reform inside-out. How can you separate the right-side out membranes from the inside-out membranes? 4. Using PubMed, find one current example of the use of lectins in research on mammalian cells. Briefly deseribe the research and give the proper citation for the article. 5.

Answer 1

3.b. Matured red blood cells do not have any organelles and other internal membrane structure. Hence pure form of plasma membrane can be obtained from red blood cells. Moreover, red blood cells do not have extensive amount of extracellular matrix attached, so working only with cell membrane is easy. For these reasons red blood cells have been used in this experiment.

4. Red blood cells were lysed and they were allowed to reform. For this some membrane will reform as inside out. This can be readily separated from the right-side out membrane. Since the phospholipid bilayer is asymmetric in nature, for the inside out membrane some of the phospholipids that should be present inside originally, will stay outside. So if a fluorescent dye or enzyme linked dye can bind a specific phospholipid only when it is outside, it can be viewed as the experiment will be spectrophotometric or colorimetric.

For instance, 2,4,6-trinitrobenzenesulfonate (TNBS) can probe phosphatidyl ethanolamine (PE) only when it is on extracellular side. A RBC which has inside out membrane structure will bind to TNBS as it has PE on its extracellular side not on cytosolic side. But other RBCs which has right-side out membrane structure will not bind to TNBS. To bind to PE on the cytosolic side TNBS must travel to the cytosolic side but TNBS can not penetrate the cell membrane. Hence the RBCs with inside out and right-side out membrane can be easily distinguished.