Ziegelhöffer A, Breier A, Dzurba A, Vrbjar N
Gen Physiol Biophys. 1983 Dec;2(6):447-56.
Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol.
异硫氰酸盐是巯基的强效修饰剂，已成功应用于研究肾（Na⁺ + K⁺）-ATP酶的活性位点结构。然而，关于异硫氰酸盐与心肌肌膜ATP酶的相互作用却知之甚少。在本研究中，研究了对溴苯基异硫氰酸盐（BPITC）对未表现出（Mg-Ca）-ATP酶活性的离体大鼠心脏肌膜制剂ATP酶活性的相互作用模式和抑制作用。浓度为10⁻⁷ - 10⁻⁴ mol·l⁻¹的BPITC选择性地、非竞争性地抑制肌膜中的（Na⁺ + K⁺）-ATP酶活性，半数抑制浓度（ID50）约为2×10⁻⁷ mol·l⁻¹。通过使膜与BPITC预孵育，保持抑制剂与膜蛋白浓度的比例恒定，消除了BPITC与反应体系中二价阳离子（即Mg²⁺和Ca²⁺）的非特异性相互作用。在这些条件下，未观察到对肌膜Mg²⁺-ATP酶或低亲和力Ca²⁺-ATP酶有明显的抑制作用。用2 mmol·l⁻¹ ATP对膜进行预孵育可保护（Na⁺ + K⁺）-ATP酶活性免受BPITC的抑制。在β-巯基乙醇或二硫苏糖醇存在下，BIPTC与肌膜的相互作用被证明是可逆的。
