Argaman A, Shoshan-Barmatz V
Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel.
J Biol Chem. 1988 May 5;263(13):6315-21.
Dicyclohexylcarbodiimide (DCCD), a hydrophobic carboxyl reagent, inhibited Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles, induced by elevated pH, tetraphenylboron, ATP + Pi, 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.
二环己基碳二亚胺（DCCD）是一种疏水性羧基试剂，它能抑制因pH升高、四苯硼、ATP + 无机磷酸或用乙酸酐进行膜修饰而诱导的、从负载Ca2+的肌浆网囊泡中释放Ca2+的过程。在所使用的条件下，抑制Ca2+释放、Ca2+-ATP酶活性和Ca2+摄取需要相同浓度的DCCD。另一方面，游离Ca2+或碱性pH可防止DCCD对Ca2+-ATP酶和偶联的Ca2+转运的抑制，但不能防止对Ca2+释放的抑制。此外，几种亲水性羧基试剂可抑制Ca2+-ATP酶，但不抑制Ca2+释放。我们认为，位于蛋白质疏水区域的一个或多个羧基残基参与了Ca2+释放的控制，DCCD与该基团的相互作用会阻断Ca2+释放。这个基团与参与抑制Ca2+-ATP酶的基团不同。DCCD还抑制[3H]ryanodine与连接肌浆网膜的结合。Ca2+的存在或碱性pH仅对DCCD抑制ryanodine结合的程度有轻微影响。用[14C]DCCD孵育这些膜，除了Ca2+-ATP酶外，还导致350、170、140、53和30 kDa蛋白质的标记。本文讨论了一种或所有被DCCD标记的蛋白质在Ca2+释放和ryanodine结合中的作用。
