1. Review Figure 7-22 in your textbook and how exposed regions of membrane prote
ID: 208505 • Letter: 1
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1. Review Figure 7-22 in your textbook and how exposed regions of membrane proteins can be labeled with i by the lactoperoxidase (LP) reaction. Similarly, carbohydrate side chains of membrane glycoproteins can be labeled with 'H (tritium) by oxidation of galactose groups with galactose oxidase (GO) followed by reduction with tritiated borohydride ("H-BH) *NOTE, both LP and GO are too large to penetrate into the interior of an intact cell or vesicle. Taking all of this into account explain each of the following observations made with intact erythrocytes using no more than 1 sentence for each answer. Think about the orientation/composition of the protein(s) in the membrane. (Note, you should review the "Key Technique" describing SDS-PAGE on pp. 172-173)(1 pt each) a) When intact cells are incubated with LP in the presence of 11 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 'H-BHa, several of the bands on the SDS-PAGE 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 GOPH-BH4 method d) None of the proteins of the plasma membrane that are known to be devoid of carbohydrate are labeled by the LP/151 method. e) If the erythrocytes are ruptured before the labeling procedure, the LP procedure labels virtually all of the major membrane proteinsExplanation / Answer
a) Membrane proteins are often flanked by aromatic residues like tyrosine oriented interfacially between the membrane-water interface. Since LP radiolables protein at tyrosine residues therefore it can be concluded that these interfacial tyrosines get radiolabelled and give band in the gel.
b) The external surface of erythrocytes contains exposed galactosyl and N-acetylgalactosaminyl residues. Since they form a component of glycoproteins thus upon exposure to GO they get radiolabelled and give bands in the gel.
c) The GO/3H-BH4 method can radiolabelled any exposed galactosyl and N-acetylgalactosaminyl residues which form a part of membrane glycolipid and glycoprotein. Therefore, all of them get radilabelled.
d) COULD NOT UNDERSTAND THE QUESTION..SORRY!!
e) When the membrane is perturbed the LP enzyme gets access to all the tyrosines in all of the membrane protiens. This is usually prevented in the normal cases as LP cannot permeate through intact plasma membrane.
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