McNicoll N, Escher E, Wilkes B C, Schiller P W, Ong H, De Léan A
Clinical Research Institute of Montreal, Canada.
Biochemistry. 1992 May 12;31(18):4487-93. doi: 10.1021/bi00133a015.
A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.
为了研究受体蛋白的肽结合结构域，并更好地表征结合位点密度低的组织中的该受体，开发了一种高效的心房利钠因子（ANF）光亲和衍生物。光敏残基的位置是根据分子构象模型和构效关系研究来选择的，这两者都表明该肽的羧基末端是疏水极的一部分，可能会在受体的ANF结合口袋内进行深度相互作用。选择光反应性残基对苯甲酰苯丙氨酸（BPA）作为肽序列第125位精氨酸的替代物，得到了一种与受体蛋白共价结合效率高（63%）的光亲和衍生物。与ANF相比，这种衍生物（BPA-ANF）的亲和力低10倍，但它在刺激牛肾上腺球状带细胞产生cGMP和抑制醛固酮分泌方面是一种完全激动剂。用BPA-ANF进行光亲和标记能特异性地识别ANF-R1和ANF-R2受体蛋白，其效率比用ANF的叠氮衍生物或交联剂高10倍。因此，这种新的ANF衍生物似乎可用于研究低表达水平组织中的ANF受体、定位细胞内化后的受体，以及标记适合氨基酸微量测序的受体蛋白水解片段。
