Dicyclohexylcarbodiimide inhibits the monoamine carrier of bovine chromaffin granule membrane

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Abstract
The monoamine carrier of bovine chromaffin granule membrane catalyzes a H+/neutral amine antiport. Dicyclohexylcarbodiimide (DCCD) inhibits this carrier in a time- and concentration-dependent manner as shown by the following evidence: it inhibits the carrier-mediated pH gradient driven monoamine uptake without collapsing the pH gradient; it affects the binding of the specific inhibitors [2-3H]dihydrotetrabenazine and [3H]reserpine. The DCCD inhibition of the carrier occurs in the same concentration range as that of the ATP-dependent H+ translocase. Saturation isotherms of [2-3H]dihydrotetrabenazine binding indicate that DCCD decreases the number of binding sites without any change of the equilibrium dissociation constant. Kinetic studies of DCCD inactivation indicate that the modification of only 1 amino acid residue is responsible for the inhibition. Preincubation of the membranes with tetrabenazine protects the carrier against inactivation by DCCD: in this case, [2-3H]dihydrotetrabenazine binding and pH gradient driven monoamine uptake are restored after washing out of DCCD and tetrabenazine. In the monoamine carrier, there may be a carboxylic acid involved in H+ translocation, similar to those demonstrated not only in F0-F1 ATPase but also in cytochrome c oxidase, mitochondrial cytochrome b-c1 complex and nucleotide transhydrogenase. Protonation-deprotonation of this group would affect the binding of [2-3H]dihydrotetrabenazine by the carrier.