He X, Nishio K, Misono K S
Department of Molecular Cardiology, Cleveland Clinic Foundation Research Institute, Ohio 44195-5071, USA.
Bioconjug Chem. 1995 Sep-Oct;6(5):541-8. doi: 10.1021/bc00035a007.
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 alpha-amino group of the ANF(4-28) peptide, and the peptide derivatives were radioiodinated at Tyr-28 to obtain affinity reagents, N4alpha-IAc-[125I]ANF(4-28), N4alpha-BrAc-[125I]ANF(4-28), and N4alpha-(maleimidobenzoyl)-[125I]ANF(4-28). Receptor labeling was carried out in a stepwise fashion as follows: (1) Membranes were treated with p-chloromercuriobenzenesulfonic acid (PCMBS) or N-ethylmaleimide to block sulfhydryl groups; (2) the affinity reagent was allowed to bind to the receptor at 0 degrees 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, the labeling 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)-[125I]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-fold greater than that by direct cross-linking using disuccinimidylsuberate and [125I]ANF(4-28) under comparable conditions. Digestion of the membrane protein labeled with IAc-[125I]ANF(4-28) by BrCN, 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.
为便于对心房利钠因子（ANF）受体进行表征，我们开发了一种亲和标记程序，即逐步亲和标记法，该方法可高效地特异性标记肾上腺质膜中的ANF结合位点。将碘乙酰基（IAc-）、溴乙酰基（BrAc-）或马来酰亚胺苯甲酰基连接到ANF(4-28)肽的氨基末端α-氨基上，并将肽衍生物在Tyr-28处进行放射性碘化，以获得亲和试剂，即N4α-IAc-[125I]ANF(4-28)、N4α-BrAc-[125I]ANF(4-28)和N4α-(马来酰亚胺苯甲酰基)-[125I]ANF(4-28)。受体标记按以下步骤进行：（1）用对氯汞苯磺酸（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；（3）洗涤膜以去除未结合的试剂，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）随后进行放射自显影，显示出对130 kDa的ANF受体进行了特异性标记。根据掺入的125I放射性活度估计，与IAc-[125I]ANF(4-28)、BrAc-[125I]ANF(4-28)和(马来酰亚胺苯甲酰基)-[125I]ANF(4-28)反应的标记产率分别为70%、52%和21%。使用IAc-[125I]ANF(4-28)的逐步程序对受体进行标记的效率比使用N3Bz-[125I]ANF(4-28)进行光亲和标记的效率高27倍，比在可比条件下使用辛二酸二琥珀酰亚胺酯和[125I]ANF(4-28)进行直接交联的效率高63倍。用溴化氰、内蛋白酶Glu-C和内蛋白酶Lys-C消化用IAc-[125I]ANF(4-28)标记的膜蛋白，分别得到表观质量为40 kDa、18 kDa和29 kDa的单条放射性标记条带。对消化产物进行反相高效液相色谱分离也得到单个主峰。每个消化产物中亲和标记局限于一个主要片段，这表明交联发生在单个或有限数量的位点上。具有高交联产率和特异性的逐步亲和标记法可能有助于分析ANF受体结合位点的结构。
