Liebmann C, Bossé R, Escher E
Institute of Biochemistry and Biophysics, Biological Faculty, University of Jena, Germany.
Mol Pharmacol. 1994 Nov;46(5):949-56.
We have synthesized a potent, selective, radioiodinated bradykinin (BK) analogue with high specific radioactivity (1000-1500 Ci/mmol). The new tracer, 125I-[p-Phe5]BK, was prepared carrier-free from the corresponding nitro precursor, [p-NO2-Phe5]BK, via catalytic hydrogenation and halodediazotation. This peptide bound to guinea pig ileum membranes in a biphasic pattern, with a high affinity dissociation constant of 3 pM (Bmax = 22 fmol/mg of protein) and a low affinity dissociation constant of 192 pM (Bmax = 245 fmol/mg of protein). The kinetically determined Kd values were 2 pM and 910 pM, respectively. The properties of the new tracer and of the peptide analogues [p-iodo-Phe5]BK and [p-NO2-Phe5]BK were compared with those of [3,4-3H(N)] [2,3-prolyl]BK as label in both saturation and inhibition studies. The results indicated that [p-iodo-Phe5]BK possessed increased affinity for the high affinity site and decreased affinity for the low affinity site, relative to BK. In rat myometrial membranes, in contrast, [p-iodo-Phe5]BK failed to reveal a high affinity site and displayed reduced affinity for the low affinity site, compared with BK. The nitro precursor was a nonselective ligand with nanomolar affinity for all labeled binding sites in both membrane preparations. Measuring the influence of BK and its analogues on guanosine-5'-O-(3-[35S]thio)triphosphate binding to guinea pig ileum membranes, we showed that G proteins were separately activated via both binding sites, qualifying these sites as constituents of signal transduction pathways and, therefore, real membrane receptors. With the new tracer as label, the B2 receptor antagonists D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]BK and D-Arg0-[Hyp3,Thi5,8,D-Phe7]BK recognized both binding sites with very high affinity in guinea pig ileum membranes, classifying these sites as B2 receptors. The BK-induced contraction in guinea pig ileum is obviously mediated via the receptor with nanomolar affinity, but the physiological role of the high affinity receptor is still unknown.
我们合成了一种具有高比放射性（1000 - 1500 Ci/mmol）的强效、选择性放射性碘化缓激肽（BK）类似物。新的示踪剂125I - [p - Phe5]BK是通过催化氢化和卤代重氮化反应，从相应的硝基前体[p - NO2 - Phe5]BK无载体地制备得到的。该肽以双相模式与豚鼠回肠膜结合，高亲和力解离常数为3 pM（Bmax = 22 fmol/mg蛋白质），低亲和力解离常数为192 pM（Bmax = 245 fmol/mg蛋白质）。动力学测定的Kd值分别为2 pM和910 pM。在饱和和抑制研究中，将新示踪剂以及肽类似物[p - 碘代 - Phe5]BK和[p - NO2 - Phe5]BK的性质与[3,4 - 3H(N)] [2,3 - 脯氨酰]BK作为标记物的性质进行了比较。结果表明，相对于BK，[p - 碘代 - Phe5]BK对高亲和力位点的亲和力增加，对低亲和力位点的亲和力降低。相比之下，在大鼠子宫肌层膜中，[p - 碘代 - Phe5]BK未显示出高亲和力位点，且与BK相比，对低亲和力位点的亲和力降低。硝基前体是一种对两种膜制剂中所有标记结合位点具有纳摩尔亲和力的非选择性配体。通过测量BK及其类似物对豚鼠回肠膜上鸟苷 - 5'-O-(3 - [35S]硫代)三磷酸结合的影响，我们表明G蛋白通过两个结合位点分别被激活，这证明这些位点是信号转导途径的组成部分，因此是真正的膜受体。以新示踪剂为标记物，B2受体拮抗剂D - Arg0 - [Hyp3,Thi5,D - Tic7,Oic8]BK和D - Arg0 - [Hyp3,Thi5,8,D - Phe7]BK在豚鼠回肠膜中以非常高的亲和力识别两个结合位点，将这些位点归类为B2受体。BK诱导的豚鼠回肠收缩显然是通过具有纳摩尔亲和力的受体介导的，但高亲和力受体的生理作用仍然未知。
