Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.
Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.
复制标题
铕标记的表皮生长因子和神经降压素：用于受体结合研究的新型探针。
作者：
O. Mazor;M. Hillairet de Boisferon;A. Lombet;A. Gruaz‐Guyon;B. Gayer;Delphine Skrzydelsky;F. Kohen;P. Forgez;A. Scherz;W. Rostène;Y. Salomon
我们研究了标记两个生物活性肽，表皮生长因子（EGF）和神经降压素（NT），与铕（Eu）-二乙烯三胺五乙酸的可能性。更具体地说，我们测试了它们作为探针在研究受体结合使用时间分辨荧光的Eu 3+。相对简单的合成产生具有类似于同位素标记的衍生物的可接受的结合特性的配体。标记的Eu-EGF与人A431表皮类癌细胞的结合亲和力（Kd）为3.6 +/- 1.2 nM，与报道的EGF的Kd值相似，而Eu-NT对人HT 29结肠癌细胞的Kd值（7.4 +/- 0.5 nM）或转染有高亲和力NT受体（CHO-NT 1）的中国仓鼠卵巢（CHO）细胞的表达量为约10- 100 nM。比NT的Kd值高1倍。通过刺激培养的小鼠D1造血细胞增殖测定的Eu标记的EGF的生物活性与用天然EGF获得的生物活性几乎相同。在CHO-NT 1细胞中，NT和Eu-NT对Ca 2+内流的最大刺激相似，但各自的K0.5值分别为20 pM和1 nM，对应于先前描述的结合亲和力的差异。这些研究的结果表明，Eu标记的肽激素和生长因子分子范围从10（3）至10（5）Da可以方便地完成。重要的是，与生物危害性放射性配体相比，Eu标记的产品稳定约2年，并且对于实验室使用是完全安全的。因此，Eu-标记的肽提出了一个有吸引力的替代常用的放射性标记的配体在生物学研究中，一般和受体测定，特别是。
We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.
DOI：
10.1073/pnas.80.5.1337
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
KAWAMOTO, T;SATO, JD;MENDELSOHN, J
通讯作者：
MENDELSOHN, J