8. Membranes grow by expansion as new protein and phospholipid components are in
ID: 205803 • Letter: 8
Question
8. Membranes grow by expansion as new protein and phospholipid components are inserted. Most proteins are inserted as they are synthesized. Phospholipids are synthesized within the plasma membrane by integral membrane proteins (enzymes) of prokaryotes. To determine whether new phospholipids are synthesized in one or both monolayers of the membrane, researchers made use of trinitrobenzenesulfonic acid (TNBS) that reacts specifically with the head group of phosphatidyl- ethanolamine (PE). Because this reagent does not penetrate membranes, it only labels external PE In the bacterium Bacillus megaterim, TNBS labeling of intact cells shows that 30% of membrane PE is in the external monolayer and 70% is in the cytoplasmic monolayer (and thus not labeled; see figure below). You use this technique to examine the distribution of PE synthesized during a 1- minute pulse of P-labeled inorganic phosphate which is incorporated into the phospholipid heads. TNBS was added either immediately after the radioactivity or 30 minutes later. The results are shown in the right portion of the figure below. (3+2+2 points) a) Is new PE synthesized in one or both monolayers of the membrane? If only one monolayer, which one? Explain, using specific terms correctly b) Given the negligible rate of spontaneous phospholipid flipping on this time scale, explain the different distribution of newly-synthesized PE immediately after the pulse and 30 minutes later. c) Phosphatidylserine in red blood cells differs from these results in that it appears to reside exclusively in the cytoplasmic monolayer. How would you explain this observation? Total PE Chromatographic separation of PE and TNBS-tagged PE Tagged with Tagged with30 min (a) Total PE isolated from intact cells after reaction with excess TNBS. (b) Pulse- labeled PE reacted immediately with TNBS or after a delay of 30 min. TNBS Not tagged Not taggedExplanation / Answer
A. The newly synthesized PE occurs in one monolayer only. The cytoplasmic layer the PE is synthesized. In figure A. the TNBS tageed-PE band is less compare to non tagged PE. TNBS binds only to the external PE and their composition is very low. So, the non tagged in cytoplamic surface and their conentration is very high. This leads to
assume that the PE synthesised in the cytoplamic side of PM.
B. Pulse labeled experiments at 0 min 32-P labelled inorganic phosphate is rich in cytoplamic side of PM. TNBS taged with 32-P phosphate is nil. All synthesized PE are in cytoplasmic side of PM. At 30 min by using flipping mechanism some 32-P PE flips to external side of PM and it binds with TNBS.
C. Phospatidylserine always present only inside the cytoplamic monolayer because it is used in apoptosis signal mechanism. If Phospatidylserine flip and moves to external PM the cell under goes apoptosis.
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