Interaction of dibucaine with the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.
Interaction of dibucaine with the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.
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地布卡因与肌浆网 Ca(2)-ATP 酶跨膜结构域的相互作用。
DOI：
10.1021/bi00206a035
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Lepock,JR
中科院分区：
文献类型：
--
作者：
Anteneodo,C;Rodahl,AM;Meiering,E;Heynen,ML;Sennisterra,GA;Lepock,JR
摘要：通过测定地布卡因对跨膜区和水相区变性的影响，研究了地布卡因与兔肌浆网Ca 2 +-ATP酶（一种离子转运膜蛋白）相互作用的位点，所述跨膜区和水相区分别含有高亲和力Ca 2+结合位点和ATP水解位点。在无Ca ~（2+）存在下，用差示扫描量热法（DSC）观察到Ca ~（2+）-ATP酶的一个不可逆变性转变，rm=49 C。在Ca 2+存在下，而不是Mg 2+、Sr 2+或Ba 2+存在下，观察到一种新的高温转变，该转变已被证明是由于跨膜区的稳定化[Lepock，J.R.，Rodahl，A. M.，张，C.，Heynen，M. L.，沃茨，B，和Cheng，KH（1990）Biochemistry 29，681 -689]。最大稳定性对应于13.8 C的Tm偏移，Hill分析表明产生稳定性的Ca 2+结合位点具有K = 2.5 X 10-4 M，协同性（n）为1。因此，稳定化是由于Ca 2+不是结合在高亲和力位点上，而是结合在先前观察到的低或中等亲和力位点上，该位点必须位于跨膜或茎亚结构域。地布卡因对水相结构域的Tm几乎没有影响，但它降低了跨膜结构域的Tm，s为4.1 X 10-4 M，协同性约为1.6，这意味着不稳定是由于地布卡因与中等或中等高亲和力的位点结合。这些位点可能位于Ca ~（2+）-ATP酶上，也可能位于脂质-蛋白质界面上。跨膜结构域Tm的降低是由于天然状态的不稳定、变性状态的稳定或不可逆变性步骤速率的增加。这些结果使用DSC是一致的敏化热失活的Ca 2+摄取的地布卡因，但缺乏对ATP水解的热失活的影响。因此，局部麻醉剂如地布卡因与整合膜蛋白相互作用的一般位点可以是通过跨膜区。
Revised Manuscript Received July 25, 1994® abstract: The site of interaction of dibucaine with the Ca2+-ATPase of rabbit sarcoplasmic reticulum, an ion-transporting membrane protein, was investigated by determining the effect of dibucaine on the denaturation of the transmembrane domain and theaqueous domain containing, respectively, the high-affinity Ca2+ binding sites andthe site of ATP hydrolysis. In the absence of Ca2+, a single irreversible denaturation transition with rm=49 Cis observed for theCa2+-ATPase by differential scanning calorimetry (DSC). In the presence of Ca2+, but notMg2+, Sr2+, or Ba2+, a new high-temperature transition is observed that has been shownto be due to stabilization of the transmembrane region [Lepock, J. R., Rodahl, A. M., Zhang, C., Heynen, M. L., Waters, B., & Cheng, KH (1990) Biochemistry 29,681-689]. The maximum stabilization corresponds to a shift in Tm of 13.8 C, and Hill analysis indicates that the Ca2+ binding site yielding stabilization has a K¿= 2.5 X 10-4 M with a cooperativity (n) of 1. Thus, stabilization is due to Ca2+ binding notto the high-affinity sites but to one of the previously observed sites of low or intermediate affinity, which must be located in the transmembrane or stalk subdomains. Dibucaine has little effect on the Tm of the aqueous domain, but it decreases the Tm of the transmembrane domain with s 4.1 X 10-4 M and a cooperativity of approximately 1.6, implying that destabilization is due to the binding of dibucaine to sites of intermediate or moderately high affinity. These sites could be located on the Ca2+-ATPase or possibly at the lipid-protein interface. The decrease in Tm of the transmembrane domain is dueto either destabilization of the native state, stabilization of the denatured state, or an increase in therate of the irreversible step of denaturation. These results using DSC are consistent with the sensitization to thermal inactivation of Ca2+ uptake by dibucaine but a lack of effect on the thermal inactivation of ATPhydrolysis. Thus, a general site of interaction of local anaesthetics such as dibucaine with integral membrane proteins may be through the transmembrane region.