The difference in rate of loss of the esr signals is

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The difference in rate of loss of the ESR signals is due to the location of the nitroxide radical on the two phospholipids. The nitroxide radical in phos- pholipid 1 is on the head group and is therefore in direct contact with the external medium. Thus, it can react quickly with ascorbate. The nitroxide radical in phospholipid 2 is attached to a fatty acid chain and is therefore H H H C C R 1 O O C C R 2 H H C O P O O - O O O choline A 1 A 2 A 1 A 2 C C D D Figure 10–13 Cleavage specificity of several phospholipases ( Answer 10–27 ). Susceptible bonds are indicated by arrows . Letters indicate specific phospholipases: A 1 is phospholipase A 1 ; A 2 is phospholipase A 2 ; C is phospholipase C; and D is phospholipase D.
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THE LIPID BILAYER A225 partially buried in the interior of the membrane. As a consequence, it is less accessible to ascorbate and is reduced more slowly. B. The key observation is that the extent of loss of ESR signal in the presence and absence of ascorbate is the same in resealed red cell ghosts, but differ- ent in red cells. These results suggest that there is an undefined reducing agent in the cytoplasm of red cells (which is absent from red cell ghosts). Like ascorbate, this cytoplasmic agent can reduce the more exposed phos- pholipid 1, but not the less exposed phospholipid 2. Thus, in red cells, phos- pholipid 2 is stable in the absence of ascorbate; in the presence of ascorbate, the spin-labeled phospholipids in the outer monolayer are reduced, causing loss of half the ESR signal. Phospholipid 1, on the other hand, is not stable in red cells in the absence of ascorbate because the phospholipids in the cyto- plasmic monolayer are exposed to the cytoplasmic reducing agent, which destroys half the ESR signal. When ascorbate is added, labeled phospho- lipids in the outer monolayer are also reduced, causing loss of the remaining ESR signal. C. The results in Figure 10–4 indicate that the labeled phospholipids were introduced equally into the two monolayers of the red cell plasma mem- brane. Phospholipid 2 was 50% sensitive to ascorbate, indicating that half the label was present in the outer monolayer, and 50% insensitive to ascor- bate, indicating that half was present in the cytoplasmic monolayer. Simi- larly, phospholipid 1 was 50% sensitive to the cytoplasmic reducing agent and 50% sensitive to ascorbate, indicating an even distribution between the cytoplasmic and outer monolayers. Reference: Rousselet A, Guthmann C, Matricon J, Bienvenue A & Devaux PF (1976) Study of the transverse diffusion of spin labeled phospholipids in bio- logical membranes: 1. Human red blood cells. Biochim. Biophys. Acta 426, 357–371. 10–29 A. Only phosphatidylserine and phosphatidylethanolamine have primary amino groups that can react with SITS. Since these phospholipids are labeled only when the red cells are made permeable (ghosts), they presum- ably reside in the cytoplasmic monolayer. This conclusion is supported by the results from experiments with sea snake venom, which degrades phos-
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