Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [125I]-alpha-bungarotoxin.

Acetylcholine receptors have been purified from junctional regions of normal rat diaphragm muscle and from extrajunctional regions of denervated diaphragm. The reaction of purified receptors with [122I]-alpha-bungarotoxin has been investigated by kinetic methods. The toxin-receptor complexes dissociated in a biphasic manner at 35 degrees with a rapidly dissociating component (t1/2 = 4 hr) and a slowly dissociating component (t1/2 is greater than or equal to 100 hr). The association reaction between toxin and receptor did not obey simple second-order kinetics but could be analyzed in terms of two classes of binding sites corresponding to the two rates of dissociation. This treatment of the data allowed derivation of association rate constants for the two sites. Value obtained for the dissociation constants were 3.7 times 10(-10) and less than or equal to 0.4 times 10(-10) M for the junctional receptor and 1.7 times 10(-10) and is less than or equal to 0.2 times 10(-10) M for the extrajunctional receptor. In each case it is the more tightly binding component that associates and dissociates more slowly. Receptors present in crude preparations were comparable to purified receptors in their reaction with [125I-alpha-bungarotoxin. The validity of the two site model is discussed in relation to the kinetic studies.