Dicyclohexylcarbodiimide interaction with sarcoplasmic reticulum. Inhibition of Ca²⁺ efflux

Dicyclohexylcarbodiimide (DCCD), a hydrophobic carboxyl reagent, inhibited Ca²⁺ release from Ca²⁺-loaded sarcoplasmic reticulum vesicles, induced by elevated pH, tetraphenylboron, ATP + P(i), or membrane modification with acetic anhydride. Under the conditions used, the same concentrations of DCCD were required for inhibition of Ca²⁺ release, Ca²⁺-ATPase activity, and Ca²⁺ uptake. On the other hand, free Ca²⁺ or alkaline pH prevented the inhibition by DCCD of Ca²⁺-ATPase and coupled Ca²⁺ transport but not that of Ca²⁺ release. Moreover, several hydrophilic carboxyl reagents inhibited Ca²⁺-ATPase but not Ca²⁺ release. We suggest that a carboxyl residue(s), located in a hydrophobic region of a protein(s), is involved in the control of Ca²⁺ release, where DCCD interaction with this group blocks Ca²⁺ release. This group is distinct from the one involved in the inhibition of Ca²⁺-ATPase. DCCD also inhibited [³H]ryanodine binding to junctional sarcoplasmic reticulum membranes. The presence of Ca²⁺ or an alkaline pH only slightly affects the degree of inhibition of ryanodine binding by DCCD. Incubation of the membranes with [¹⁴C]DCCD resulted in labeling of 350-, 170-, 140-, 53-, and 30-kDa proteins in addition to the Ca²⁺-ATPase. The involvement of one or all of the DCCD-labeled proteins in Ca²⁺ release and ryanodine binding is discussed.