Larose L, McNicoll N, Rondeau J J, Escher E, De Lean A
Laboratory of Molecular Pharmacology, Clinical Research Institute of Montreal, Canada.
Biochem J. 1990 Apr 15;267(2):379-84. doi: 10.1042/bj2670379.
In bovine adrenal zona glomerulosa, atrial natriuretic factor (ANF) exerts its physiological effect through high-affinity binding to specific membrane receptors. On studying further the molecular properties of the ANF receptor binding domain, we have observed that incubation of intact or solubilized bovine adrenal zona glomerulosa membranes with 125I-ANF-(99-126) followed by u.v. irradiation results in the irreversible labelling of a 130 kDa protein corresponding to the ANF-RI receptor. This process is time-, protein- and 125I-ANF-dependent. The apparently covalent nature of this complex is documented by its resistance to heat, guanidine hydrochloride, urea and trichloroacetic acid denaturation. Photolabelling with underivatized 125I-ANF is much more efficient with the ANF-R1 than with the ANF-R2 receptor. After photolysis, the covalently linked 125I-ANF is still sensitive to digestion by carboxypeptidase A, suggesting that ANF is linked by its N-terminal end to the receptor upon u.v. irradiation and that its C-terminal end is still freely accessible. Aerobic conditions and lipids are required for the photolabelling, suggesting a role in this process for malondialdehyde, a highly reactive secondary product associated with u.v.-induced lipid peroxidation. This simple method should provide a powerful tool in the accurate characterization of the hormone-binding domain of the ANF receptor.
在牛肾上腺球状带中，心钠素（ANF）通过与特定膜受体的高亲和力结合发挥其生理作用。在进一步研究ANF受体结合域的分子特性时，我们观察到，将完整的或可溶解的牛肾上腺球状带膜与125I-ANF-(99-126)一起孵育，随后进行紫外线照射，会导致一种与ANF-RI受体相对应的130 kDa蛋白质发生不可逆标记。这个过程是时间、蛋白质和125I-ANF依赖性的。这种复合物明显的共价性质通过其对热、盐酸胍、尿素和三氯乙酸变性的抗性得以证明。未衍生化的125I-ANF对ANF-R1的光标记比对ANF-R2受体更有效。光解后，共价连接的125I-ANF仍然对羧肽酶A的消化敏感，这表明在紫外线照射下，ANF通过其N末端与受体相连，而其C末端仍然可以自由接近。光标记需要有氧条件和脂质，这表明与紫外线诱导的脂质过氧化相关的高反应性二级产物丙二醛在这个过程中起作用。这种简单的方法应该为准确表征ANF受体的激素结合域提供一个有力的工具。
