Phatidylserine and only in the permeable ghosts these

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phatidylserine and phosphatidylethanolamine only in the permeable ghosts. These results, taken together, indicate that the phosphatidylserine and phosphatidylethanolamine are localized almost exclusively in the cyto- plasmic monolayer of red cell membranes. Phospholipase degradation of phosphatidylcholine and sphingomyelin in intact red cells indicates that they are present in the outer monolayer. This conclusion depends on the red cells remaining intact during the treatment. In the case of sea snake venom, the absence of degradation of phos- phatidylserine and phosphatidylethanolamine in intact red cells provides an internal control. In the case of sphingomyelinase, there is no internal control, but the absence of lysis indicates that the membrane is intact. The results in Table 10–2 do not exclude the possibility that phos- phatidylcholine and sphingomyelin are also located in the cytoplasmic monolayer. Since most of the sphingomyelin is degraded by sphin- gomyelinase, most of it must be localized in the outer monolayer of the membrane. No such data are provided for phosphatidylcholine; thus, it would be incorrect to conclude that phosphatidylcholine is located exclu- sively in the outer monolayer. Other experiments not reported here do suggest, however, that phosphatidylcholine is found almost entirely in the outer monolayer. B. You chose red cells for these experiments because they contain no internal membranes. If the same experiments were performed on cells with internal membranes, it would have been impossible to measure the phospholipid composition of the cytoplasmic monolayer directly, since phospholipids from the cytoplasmic monolayer would have been hopelessly confused with those from internal membranes.
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A226 Chapter 10: Membrane Structure References : Bretscher M (1972) Asymmetrical lipid bilayer structure for bio- logical membranes. Nat. New Biol. 236, 11–12. Deenen LLM & DeGier J (1974) Lipids of the red cell membrane. In The Red Blood Cell (D. MacN. Surgenor, ed), pp 147–211. New York: Academic Press. 10–30 A. PLAP is soluble in ice-cold Triton X-100 until 20 to 40 minutes after its synth- esis. This time delay, coupled with the shift in mobility by SDS-gel elec- trophoresis, suggests that at 20 to 40 minutes PLAP is converted to its GPI- anchored form, which migrates differently from the unmodified form. This is consistent with what is known about synthesis of GPI-anchored proteins: in the ER they are cleaved from a transmembrane anchor and attached to GPI. B. When fully solubilized in octyl glucoside, PLAP bands at the bottom of the sucrose density gradient, which is at the density characteristic of proteins. In its insoluble Triton X-100 form, PLAP bands at a less dense position, indi- cating it is complexed with molecules such as lipids, which are much less dense than proteins.
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