Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C.
Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C.
复制标题
三氟拉嗪与猪脑钙调蛋白和骨骼肌肌钙蛋白结合 C.
DOI：
10.1021/bi00455a012
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Jarrett,HW
中科院分区：
文献类型：
--
作者：
Massom,L;Lee,H;Jarrett,HW
1989年9月1日收到的修订稿件摘要：用分子筛柱自动高效液相色谱结合分析法测定了吩噻嗪镇静剂三氟拉嗪(TFP)与猪脑钙调蛋白(CaM)和兔骨骼肌肌钙蛋白C(TNC)的结合；每次注射10罐蛋白质足以确定TFP结合，结果与平衡透析获得的结果相当。在无钙时，两种蛋白几乎不能结合，而在有钙时，两种蛋白都能与4当量的TFP结合。然而，TFP结合的其他特征对于每种蛋白质来说是不同的。CaM的半峰结合出现在5.8µTFP处，Hill系数为0.82，与Scatchard方程的拟合符合4个独立的TFP结合位点。一个蜂毒素的结合取代了CaM中的两个TFP。因此，有两类可识别的TFP结合位点：被蜂毒素取代的和不被蜂毒素取代的。TFP使CaM的钙亲和力增加，必须有三个Ca~(2+)与CaM结合才能发生TFP结合。这些研究还得出了CaM的三个钙结合位点的内在亲和力的测量结果，这与以前的报道一致。肌钙蛋白C的半峰结合峰出现在16µTFP处，Hill系数为1.7，四个结合位点的数据均符合Adair方程。在1 mM的Ca~(2+)中，测得的常数Ku_2、K_3和K_4分别为2.5×104、6.6×103、5.8×105和2.0×105M~(-1)，当加入镁离子时，K_(K2)、K_3和K_4相近。TFP还会增加肌钙蛋白C的钙亲和力，受影响的是低亲和力的钙离子特异性结合位点。这些研究得出了这些钙结合位点的内在亲和力的测量结果，这与以前的测量结果一致。(细胞内钙离子浓度的变化经常会对细胞的新陈代谢产生深远的影响。钙离子在调节肌肉收缩、神经传递、分泌和其他基本生物过程中的作用早已被人们所认识。钙离子的这些作用通常被发现是由一类同源的钙结合蛋白介导的，其中包括肌钙蛋白C和钙调蛋白。CaM1是一种依赖于钙离子的多种酶的激活剂，而TnC是一种结构相似的蛋白质，适用于调节心肌和骨骼肌收缩的更专门的角色。TFP是一种吩噻嗪镇静剂，是一种微摩尔浓度的CaM拮抗剂(Weiss&Levin，1978)。后来，人们发现TFP也与Tn C结合(Levin&Weiss，1978)。其他吩噻嗪(如氯丙嗪和氟吩嗪)(Levin&Weiss，1978；Silver等人，1986)、一系列萘磺酰胺衍生物(Hidaka等人，1980)和染料Calmidazolium(Silver等人，1986)也被TNC和CaM两者结合并影响其生物活性。TFP和许多其他化合物增加了肌丝收缩的钙敏感性，并且对收缩能力的影响与这些化合物中的TnC结合很好地相关(Herzig等人，1987)。Levin和Weiss(1978)和Marshak等人(1985)都测量了吩噻嗪与CaM的结合，并对结合位点的数量和结合的钙离子依赖性得出了截然不同的结论。这种差异以及TnC上药物结合位点数量和亲和力的不确定性导致我们重新研究药物与这两种蛋白质的结合。
Revised Manuscript Received September 1, 1989 abstract: Binding of trifluoperazine (TFP), a phenothiazine tranquilizer, to porcine brain calmodulin (CaM) and rabbit skeletal muscle troponin C (Tn C) was measured by an automated high-performance liquid chromatography binding assay using a molecular sieving column; 10 jug of either protein per injection is sufficient for determining TFP binding, and results are comparable to those obtained by equilibrium dialysis. Very little binding was observed toeither protein in the absence of Ca2+ while in the presence of Ca2+ both proteins bind 4 equiv of TFP. Other characteristics of TFP binding however are different for each protein. For CaM, half-maximal binding occurs at 5.8 µ TFP, the Hill coefficient is 0.82, and the fit of the data to the Scatchard equation is consistent with four independent TFP-binding sites. Binding of one melittin displaces two TFP from CaM. Thus, there are two recognizable classes of TFP-binding sites: those that are displaced by melittin and those that are not. TFP causes an increase in the Ca2+ affinity of CaM, and three Ca2+ must be bound to CaM for TFP binding to occur. The studies also yielded a measure of the intrinsic affinity of three of CaM’s Ca2+-binding sites that is in agreement with previous reports. For troponin C, half-maximal binding occurs at 16 µ TFP, the Hill coefficient is 1.7, and the data best fit the Adair equation for four binding sites. The measured constants Ku K2, K3, and K4 were 2.5 X 104, 6.6 X 103, 5.8 X 105, and 2.0 X 105 M" 1, respectively, in 1 mM Ca2+ and were similar when Mg2+ was additionally included. TFP also increases troponin C’s Ca2+ affinity, and it is the low-affinity, Ca2+-specific binding sites that are affected. These studies yielded a measure of the intrinsic affinity of these Ca2+-binding sites that is in agreement with previous measurements.(Changes in intracellular Ca2+ concentration frequently cause profound effects upon a cell’s metabolism. The role of Ca2+ in the regulation of muscle contraction, nerve transmission, secretion, and other basic biological processes has long been appreciated. These effects of Ca2+ have often been found to be mediated by a class of homologous Ca2+-binding proteins that includes troponin C and calmodulin. CaM1 is a Ca2+-dependent activator of numerous enzymes while Tn C is a structurally similar protein adapted to the more specialized role of regulating cardiac and skeletal muscle contraction. TFP, a phenothiazine tranquilizer, is a CaM antagonist effective at micromolar concentrations (Weiss & Levin, 1978). Later, it was discovered that TFP also binds toTn C (Levin & Weiss, 1978). Other phenothiazines (eg, chlorpromazine and fluphenazine)(Levin & Weiss, 1978; Silver et al., 1986), a series of naphthylsulfonamide derivatives (Hidaka et al., 1980), and the dye calmidazolium (Silver et al., 1986) are also bound by and effect the biological activity of both Tn C and CaM. TFP and many of these other compounds increase the Ca2+ sensitivity of myofilament contraction, and the effect on contractility correlates well with Tn C binding across a series of these compounds (Herzig et al., 1987). Levin and Weiss (1978) and Marshak et al.(1985) have both measured phenothiazine binding to CaM and arrived at quite different conclusions as to the number of binding sites and the Ca2+ dependency of binding. This difference as well as the uncertainty in the number and affinity of drug-binding sites on Tn C led us to reinvestigate drug binding to both proteins.