High-yield affinity alkylation of the atrial natriuretic factor receptor binding site.
High-yield affinity alkylation of the atrial natriuretic factor receptor binding site.
复制标题
心房钠尿因子受体结合位点的高产率亲和力烷基化。
DOI：
10.1021/bc00035a007
复制
发表时间：
1995
影响因子：
4.7
通讯作者：
Misono,KS
中科院分区：
文献类型：
--
作者：
He,X;Nishio,K;Misono,KS
为了便于表征的心钠素（ANF）受体，我们已经开发了一种亲和标记程序，逐步亲和标记，它允许特异性标记的ANF结合位点在肾上腺质膜高产率。碘乙酰基（Iac-）、溴乙酰基（BrAc-）或马来酰亚氨基苯甲酰基连接到ANF（4-28）肽的氨基末端a-氨基上，并将肽衍生物在Tyr-28处进行放射性碘化，获得亲和试剂IV 4a-Iac-[125 I]-ANF（4-28）、N4 a-BrAc-[125 I] ANF（4-28）、和N4a-（马来酰亚胺苯甲酰基）-[125 I] ANF（4-28）。受体标记以如下逐步方式进行：（1）用对氯汞基苯磺酸（PCMBS）或N-乙基马来酰亚胺处理膜以封闭巯基;（2）使亲和试剂在0 ℃下与受体结合1小时;和（3）洗涤膜以除去未结合的试剂并在室温下孵育以进行烷基化反应。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE），然后放射自显影显示130 kDa的心钠素受体的特异性标记。根据掺入的125 I放射性，估计与Iac-[125 I] ANF（4-28）、BrAc-[125 I] ANF（4 - 28）和（马来酰亚胺苯甲酰基）-[126 I] ANF（4 - 28）反应的产率分别为70%、52%和21%。用Iac-[125 I] ANF（4-28）分步法标记受体的效率比用N3 Bz-[125 I] ANF（4 - 28）光亲和标记的效率高27倍，比用二琥珀酰亚胺辛二酸酯和[126 I] ANF（4-28）直接交联的效率高63倍。用BrCN、内切蛋白酶Glu-C和内切蛋白酶Lys-C消化IAc-[125 I] ANF（4-28）标记的膜蛋白，得到单个放射性标记条带，表观质量分别为40、18和29 kDa。反相高效液相色谱分离的反相也得到了单一的主峰。在每个消化物中，亲和标记局限于一个主要片段，表明交联发生在单个或有限数量的位点。具有高交联产率和特异性的逐步亲和标记可用于分析ANF受体结合位点结构。心房利钠因子（ANF）1是由心房分泌的肽激素，其具有有效的利钠作用（de Bold等人，1981）和血管舒张活性（Currie等，1983; Grammer等人，1983年）。ANF在其靶器官（包括血管、肾和肾上腺）的作用由直接偶联至鸟苷酸环化酶的细胞膜受体介导（Cantin和Genest，1985; Gerzer等人，1987年）。ANF受体分子由分子量为约130 kDa的单一多肽链组成（Kuno等人，1986; Takayanagi等人，1987; Meloche等人，1988），其含有细胞外ANF结合结构域、单一跨膜序列和含有蛋白激酶同源结构域和鸟苷酸环化酶结构域的细胞内区域（Chinkers等人，1989年）。有人认为，在基础状态下，激酶同源结构域与鸟苷酸环化酶结构域相互作用，抑制鸟苷酸环化酶活性。ANF与细胞外结构域的结合引起共形-
To facilitate characterization of the atrial natriuretic factor (ANF) receptor, we have developed an affinity labeling procedure, stepwise affinity labeling, which allows specific labeling of ANF binding sites in adrenal plasma membranes at high yields. An iodoacetyl (IAc-), bromoacetyl (BrAc-), or maleimidobenzoyl group was attached to the amino-terminal a-amino group of the ANF (4-28) peptide, and the peptide derivatives were radioiodinated at Tyr-28 to obtain affinity reagents, IV4a-IAc-[125I]-ANF (4—28), N4a-BrAc-[125I] ANF (4—28), and N4a-(maleimidobenzoyl)-[125I] ANF (4—28). Receptor label-ing was carried out in a stepwise fashion as follows:(1) Membranes were treated with p-chloromercuriobenzenesulfonic acid (PCMBS) or 7V-ethylmaleimide to block sulfhydryl groups;(2) the affinity reagent was allowed to bind to the receptor at 0 C for 1 h; and (3) the membranes were washed to remove unbound reagent and were incubated at room temperature to effect alkylation reaction. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography revealed specific labeling of a 130-kDa ANF receptor. On the basis of 125I-radioactivity incorporated, thelabeling yields were estimated to be 70%, 52%, and 21% for the reactions with IAc-[125I] ANF (4—28), BrAc-[125I] ANF (4—28), and (maleimidobenzoyl)-[126I] ANF (4—28), respectively. The efficiency of receptor labeling by the stepwise procedure using IAc-[125I] ANF (4—28) was 27-fold greater than that obtained by photoaffinity labeling using N3Bz-[125I] ANF (4—28) and 63-foldgreater than that by direct cross-linking using disuccinimidylsuberate and [126I] ANF (4-28) under comparable conditions. Digestion of the membrane proteinlabeled with IAc-[125I] ANF (4—28) byBrCN, endoproteinase Glu-C, and endoproteinase Lys-C gave single radiolabeled bands with apparent masses of 40, 18, and 29 kDa, respectively. Reversed-phase HPLC separation of the digests also gave single major peaks. The confinement of the affinity label to one major fragment in each digest suggests that the cross-linking occurred at a single or a limited number of sites. The stepwise affinity labeling with the high cross-linking yield and specificity may be useful for analyzing the ANF receptor binding site structure.Atrial natriuretic factor (ANF) 1 is a peptide hormone secreted by the heart atrium that has potent natriuretic (de Bold et al., 1981) andvasorelaxant activities (Currie et al., 1983; Grammer et al., 1983). The actions of ANF at its target organs, including the bloodvessels, kidney, and adrenal gland, are mediated by cell membrane receptors that are directly coupled to guanylate cyclase (Cantin and Genest, 1985; Gerzer et al., 1987). The ANF receptor molecule consists of a single polypeptide chain with a molecular mass of about 130 kDa (Kuno et al., 1986; Takayanagi et al., 1987; Meloche et al., 1988), containing an extracellular ANF-binding domain, a single transmembrane sequence, and an intracellular region containing both protein kinase-homologous domain and guanylate cyclase domain (Chinkers et al., 1989). It has been suggested that, in thebasal state, the kinase-homologous domain interacts with the guanylate cyclase domain, suppressing guanylate cyclase activity. Binding of ANF to the extracellular domain causes a conforma-