Effect of N, N′-dicyclohexylcarbodiimide on the binding of vesamicol, an inhibitor of acetylcholine transport into synaptic vesicles

Abstract

Vesamicol is a highly potent inhibitor of active acetylcholine transport into isolated cholinergic vesicles from Torpedo. On the basis of transport kinetics and vesamicol sensitivity, we have shown that the acetylcholine transporter could be in an activated state even in the absence of a stimulated ATPase. In this preparation, N, N′-dicyclohexylcarbodiimide (DCCD), an hydrophobic carbodiimide, inactivates both ACh transport and vesamicol binding. Inhibition of vesamicol binding by DCCD is time dependent, saturable and prevented by vesamicol. DCCD first affected the affinity constant for vesamicol. K i-value for DCCD lies in the micromolar range. These results imply that there is a DCCD reactive site within the ACh transporter and that it is located in an hydrophobic environment near the vesamicol binding site. SDS gel electrophoresis after labelling of the vesicle membrane proteins with [ 14C]DCCD shows that radioactivity is mainly incorporated in a 15 kDa subunit. Time-course and concentration dependence of [ 14C]DCCD labelling and vesamicol inhibition do not coincide. Hence, the two processes are probably unrelated and the result rather points to another inactivation mechanism which can be an intramolecluar cross link.