Chapter 7, Problem 7.11PS

Problem Set

Inside or Outside? From Figure 7-22, we know that exposed regions of membrane proteins can be labeled with ¹²⁵I by the lactoperoxidase (LP) reaction. Similarly, carbohydrate side chains of membrane glycoproteins can be labeled with 3H by oxidation of galactose groups with galactose oxidase (GO) followed by reduction with tritiated borohydride (³H—BH₄). 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 ¹²⁵I 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 ³H—BH₄, 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/³H—BH₄ method.

(d)    None of the proteins of the erythrocyte plasma membrane that are known to be devoid of carbohydrate is labeled by the LP/¹²⁵I method.

(e)    If the erythrocytes are ruptured before the labeling procedure, the LP procedure labels virtually all of the major membrane proteins.

Figure 7-22 A Method for Labeling Proteins Exposed on One or Both Surfaces of a Membrane Vesicle. (a) In the presence of ¹²⁵I, lactoperoxidase (LP) labels membrane proteins exposed on the outer surface of membrane vesicles (i.e., proteins A and B). If membrane vesicles are (b) first incubated in a hypotonic medium to make them permeable to LP and (c) then transferred to an isotonic solution containing ¹²⁵I but no external LP, the membrane proteins exposed on the inner membrane surface (i.e., proteins B and C) become labeled.