Becker's World of the Cell 9th Edition by Lewis Kleinsmith, Jeff Hardin, Gregory Paul Bertoni

Exercise 12

Inside-Out Membranes. It is technically possible to prepare sealed vesicles from erythrocyte membranes in which the original orientation of the membrane is inverted. Such vesicles have what was originally the cytoplasmic side of the membrane facing outward.
(a)What results would you expect if such inside-out vesicles were subjected to the GO/ 3 H - BH 4 procedure described in Problem 1
(b)What results would you expect if such inside-out vesicles were subjected to the LP/ 125 I procedure of Problem 1
(c)What conclusion would you draw if some of the proteins that become labeled by the LP/ 125 I method of part b were among those that had been labeled when intact cells were treated in the same way in Problem 1a
(d)Knowing that it is possible to prepare inside-out vesicles from erythrocyte plasma membranes, can you think of a way to label a transmembrane protein with 3 H on one side of the membrane and with 125 I on the other side
Problem 1
Inside or Outside From Figure 7-24, we know that exposed regions of membrane proteins can be labeled with 125 I by the lactoperoxidase (LP)reaction. Similarly, carbohydrate side chains of membrane glycoproteins can be labeled with 3 H by oxidation of galactose groups with galactose oxidase (GO)followed by reduction with tritiated borohydride ( 3 H - BH 4 ). Noting that both LP and GO are too large to penetrate into the interior of an intact cell, explain each of the following observations made with intact erythrocytes.
(a)When intact cells are incubated with LP in the presence of 125 I and the membrane proteins are then extracted and analyzed on SDS-polyacrylamide gels, several of the bands on the gel are found to be radioactive.
(b)When intact cells are incubated with GO and then reduced with 3 H - BH 4 , several of the bands on the gel are found to be radioactive.
(c)All of the proteins of the plasma membrane that are known to contain carbohydrates are labeled by the GO/ 3 H - BH 4 method.
(d)None of the proteins of the erythrocyte plasma membrane that are known to be devoid of carbohydrate is labeled by the LP/ 125 I method.
(e)If the erythrocytes are ruptured before the labeling procedure, the LP procedure labels virtually all of the major membrane proteins.