Cherifi Y, Pigeon C, Le Romancer M, Bado A, Reyl-Desmars F, Lewin M J
Unité de Recherches de Gastroentérologie, Institut National de la Santé et de la Recherche Médicale U10, Hôpital Bichat, Paris, France.
J Biol Chem. 1992 Dec 15;267(35):25315-20.
The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.
组胺H3受体激动剂（R）α-甲基组胺（MeHA）在纳摩尔浓度范围内，可抑制人胃肿瘤细胞系HGT1-克隆6中基础状态及氨甲酰胆碱刺激的肌醇磷酸生成。微摩尔浓度的组胺H3受体拮抗剂硫代哌啶可逆转这种抑制作用，且该抑制作用对霍乱毒素或百日咳毒素处理敏感。以[3H]Nα-MeHA作为特异性示踪剂，在不存在或存在50μM GTPγS的情况下，分别显示出高亲和力结合位点，其Bmax为54±3 fmol/mg蛋白质，KD分别为0.61±0.04或2.2±0.4 nM。通过Triton X-100使结合位点溶解，并通过凝胶色谱进行预纯化。通过琼脂糖-法莫替丁过滤将它们与组胺H2受体位点分离，最终保留在琼脂糖-硫代哌啶上。在SDS-聚丙烯酰胺凝胶电泳中，纯化后的位点集中在一条单一的70 kDa银染蛋白带中。它们以1.6±0.1 nM的KD和12,000±750 pmol/mg蛋白质的Bmax特异性结合[3H]Nα-MeHA。这相当于相对于细胞裂解物有90,225倍的纯化，纯度为84%。Nα-MeHA（IC50 = 5.8±0.7 nM）、（R）α-MeHA（IC50 = 9±1 nM）和硫代哌啶（IC50 = 85±10 nM）可竞争性取代结合，但法莫替丁（H2拮抗剂）或美吡拉敏（H1拮抗剂）则不能。这些发现首次为组胺H3受体蛋白的溶解、纯化及分子量表征，以及该受体通过迄今未明确的G蛋白与磷脂酰肌醇代谢的负性偶联提供了证据。
