Question

SDS-PAGE is often used to determine the location and orientation of membrane associated proteins. Commonly, experimental preparations of membranes are treated in a variety of ways in order to extract the proteins, which are then subjected to SDS-PAGE. These treatments will affect membrane proteins in different ways, based on how they are arranged in the membrane. One of the most common pre-treatments is by the digestive enzyme trypsin, which degrades polypeptide chains. Trypsin is too large to cross a membrane, but can digest exposed parts of membrane proteins. Those parts of a membrane protein that are in contact with external aqueous medium containing trypsin are digested, but the enzyme cannot digest the parts of the protein that are embedded within the lipid bilayer, or the parts of the proteins on the cytoplasmic side of the membrane if the cell is intact. If detergents are used to gently permeabilize the membrane preps prior to placing them in trypsin containing medium, the enzyme can access both sides of the membrane and will digest any polypeptide chains that is exposed and accessible. a) Draw a portion of a plasma membrane of a cell and label the cytoplasmic and extracellular surface. Place the following four proteins in the membrane: a) peripheral membrane protein (cytoplasmic exposure), b) integral protein (transmembrane exposure), c) integral protein (extracellular exposure) and d) peripheral protein (extracellular exposure) b) Describe what will happen to each of your proteins when this cell is exposed to trypsin only. c) Describe what will happen to each of your proteins when this cell is treated briefly with a detergent first, and then exposed to trypsin.

Answer 1

a)

Peripheral Protein Cast. Anbrosure Insteareal Prédion (Transme Peripheral Mehare Protein (cy to blosmic) Lipid Bilayen

b) When this cell is exposed to Trypsin only, then the peripheral protein with extracellular exposure, the transmembrane protein and the integral protein with extracellular exposure will be digested.However the degree of digestion will differ. Peripheral protein will be completely digested since none of its regions are inserted into the plasma membrane, The integral protein with extracellular exposure will have its extracellular part degraded with its membrane embedded part intact. Only the extracellular part of the transmembrane protein will be protein, with its membrane spanning segment and cytoplasmic segment intact.

c) When the cell is treated with detergent, the detergent will emulsify the lipid portion of the plasma membrane. This will lead to complelte lysis of the cell resulting in the complete release of integral and transmembrane protein. Due to this, all the protein segments previously inaccessible to Trypsin will be exposed . Thus they will be digested by Trypsin.