TY - JOUR
T1 - Affinity Labeling of Receptors
AU - Zisapel, Nava
AU - Sokolovsky, Mordechai
N1 - Funding Information:
Nicotinic acetylcholine receptors are integral proteins of the sub-synaptic membranes of skeletal muscle and of homologous electrocytes of electric fish. These receptors translate the binding of acetylcholine into an increase in the permeability of the membrane to sodium, potassium, and calcium ions. The acetylcholine binding site has been affinity labeled by a two-step procedure, the first step of which is the reduction of a disulfide group close to the acetylcholine binding site and the second, the affinity alkylation of one of the sulfhydryl groups formed by reduction. Two types of affinity alkylating agents have been used. One type, exemplified by 4-(N-maleimido) benzyltrimetylammonium iodide (MBTA), fixes the receptor in an inactive state. 2,3 A second type, exemplified by bromoacetylcholine bromide, fixes the receptor in an active state. 4 In all cases, these affinity labels act as completely reversible activators or competitive inhibitors of the unreduced receptor. Radioactively tagged MBTA has been used to quantitate receptor in intact cells, 5 in membrane fragments, 6 and in solution, 7,s and to identify the receptor subunit bear- 1This research was supported by research grants from the U.S. Public Health Service (NS07065) and from the National Science Foundation (BMS75-03026). A. Karlin and M. Winnik, Proc. Natl. Acad. Sci. U~.A. 60, 668 (1968). 3 A. Karlin, J. Gen. Physiol. 54, 245s (1969). 4 I. Silman and A. Karlin, Science 164, 1420 (1969). 5A. Karlin, J. Prives, W. Deal, and M. Winnik, J. Mol. Biol. 61, 175 (1971). ~A. Karlin and D. A. Cowburn, in "Neurochemistry of Cholinergic Receptors" (E. DeRobertis and J. Schacht, eds.), p. 37. Raven, New York, 1974. 'A. Karlin and D. A. Cowburn, Proc. Natl. Acad. Sci. U~zt. 70, 3636 (1973). s A. Karlin, M. G. McNamee, and D. A. Cowburn, Anal. Biochem. 76, 442 (1976).
PY - 1977/1/1
Y1 - 1977/1/1
N2 - The term receptor in the context used currently in drug and hormonal studies is defined operationally as those molecules that specifically recognize and bind the drug or hormone and, as a consequence of this recognition, can lead to other changes, which ultimately result in biological response. Most drug and hormone receptors that are membrane bound are in the category of “integral” membrane proteins; that is, they are not readily solubilized from the membrane and require detergents or other hydrophobie bond-breaking agents to release them. Specific labeling that may be achieved by affinity labels is often needed in order to follow changes in the quantity or distribution of certain receptors. The application of the method to the identification and quantitation of receptors has been reviewed in this chapter. It details the purification, pharmacological studies, and biochemical structure-function studies. The chapter concludes that although affinity labeling has important contributions to make in solving the complex problems of receptor structure-function relationship, the technique must be used with caution since the perturbation caused by the presence of a covalently bound ligand is capable of altering the system under study in ways that are not as yet fully understood.
AB - The term receptor in the context used currently in drug and hormonal studies is defined operationally as those molecules that specifically recognize and bind the drug or hormone and, as a consequence of this recognition, can lead to other changes, which ultimately result in biological response. Most drug and hormone receptors that are membrane bound are in the category of “integral” membrane proteins; that is, they are not readily solubilized from the membrane and require detergents or other hydrophobie bond-breaking agents to release them. Specific labeling that may be achieved by affinity labels is often needed in order to follow changes in the quantity or distribution of certain receptors. The application of the method to the identification and quantitation of receptors has been reviewed in this chapter. It details the purification, pharmacological studies, and biochemical structure-function studies. The chapter concludes that although affinity labeling has important contributions to make in solving the complex problems of receptor structure-function relationship, the technique must be used with caution since the perturbation caused by the presence of a covalently bound ligand is capable of altering the system under study in ways that are not as yet fully understood.
UR - http://www.scopus.com/inward/record.url?scp=0017338266&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(77)46071-3
DO - 10.1016/S0076-6879(77)46071-3
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AN - SCOPUS:0017338266
SN - 0076-6879
VL - 46
SP - 572
EP - 582
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -