Grover A K, Kwan C Y, Kostka P, Daniel E E
Eur J Pharmacol. 1985 Jun 7;112(2):137-43. doi: 10.1016/0014-2999(85)90489-3.
Rat mesenteric artery microsomes were previously reported to degrade 125I-angiotensin II (AII). It is now shown here that washing the membranes with EDTA and including EGTA in the assay media reduces the 125I-AII degradation to very low levels without reducing the specific binding of 125I-AII. Using EDTA wash and including 5 mM MgCl2 and 0.2 mM EGTA the following characteristics of the binding were observed: microscopic association rate constant (k1) = 1.3 to 2.2 X 10(5) M-1 s-1, microscopic dissociation rate constant (k-1) = 3.8 to 6.4 X 10(-4) s-1, equilibrium dissociation constant (Kd) = 1.8 nM and number of binding sites (Bmax) = 193 fmol/mg protein. The subcellular distribution of the specific binding of 125I-AII at 0.16 nM and 1.63 nM was studied along with the distribution of the marker enzymes. The specific binding paralleled the plasma membrane marker (5'-nucleotidase), but not the putative endoplasmic reticulum marker (NADPH-cyt. c-reductase) or the inner mitochondrial marker (cyt. c-oxidase). Thus the binding to the plasma membrane-enriched fraction F2 occurred with a similar affinity (Kd = 2.2 nM) but with higher number of binding sites (420 fmol/mg protein). This study establishes the 125I-AII binding method suitable for determining the changes in the angiotensin receptor characteristics in the pathophysiology of the vascular smooth muscle.
先前有报道称大鼠肠系膜动脉微粒体可降解125I-血管紧张素II（AII）。本文现已表明，用EDTA洗涤膜并在测定介质中加入EGTA可将125I-AII的降解降低至极低水平，而不会降低125I-AII的特异性结合。使用EDTA洗涤并加入5 mM MgCl2和0.2 mM EGTA，观察到以下结合特性：微观缔合速率常数（k1）= 1.3至2.2×10（5）M-1 s-1，微观解离速率常数（k-1）= 3.8至6.4×10（-4）s-1，平衡解离常数（Kd）= 1.8 nM，结合位点数（Bmax）= 193 fmol/mg蛋白质。研究了0.16 nM和1.63 nM时125I-AII特异性结合的亚细胞分布以及标记酶的分布。特异性结合与质膜标记物（5'-核苷酸酶）平行，但与假定的内质网标记物（NADPH-细胞色素c还原酶）或线粒体内膜标记物（细胞色素c氧化酶）不平行。因此，与富含质膜的组分F2的结合具有相似的亲和力（Kd = 2.2 nM），但结合位点数更多（420 fmol/mg蛋白质）。本研究建立了适用于确定血管平滑肌病理生理学中血管紧张素受体特性变化的125I-AII结合方法。
