Keppens S, De Wulf H, Clauser P, Jard S, Morgat J L
Biochem J. 1982 Dec 15;208(3):809-17. doi: 10.1042/bj2080809.
Specific angiotensin binding to rat hepatocytes and purified liver plasma membranes was measured by using biologically active [(3)H]angiotensin (sp. radioactivity 14Ci/mmol). The kinetic parameters for angiotensin binding to hepatocytes are: K(+1) (association rate constant). 100mum(-1).min(-1); K(-1) (dissociation rate constant), 2min(-1); K(d) (dissociation constant). 30nm; maximal binding capacity, 0.42pmol/10(6) cells or 260000 sites/cell. Angiotensin binding to membranes is profoundly affected by GTP (0.1mm) and NaCl (100mm); these regulatory compounds greatly enhance both the rate of association and of dissociation and also the extent of dissociation. K(d) amounts to 10nm in the presence of GTP+NaCl and to 1.5nm in their absence; maximal binding capacity is 0.70pmol/mg of protein, both with or without GTP+NaCl. The relative affinities of 11 angiotensin structural analogues were deduced from competition experiments for [(3)H]angiotensin binding to hepatocytes and to membranes (in the latter case, GTP + NaCl were not included, in order to study the higher affinity state of the receptor). These are highly correlated with their biological activity (activation of glycogen phosphorylase in hepatocytes). Binding to membranes occurs in the same concentration range as the biological effect. On the other hand, the existence of numerous spare receptors is suggested by the observation that binding of the agonists to hepatocytes requires 25-fold higher concentrations than those needed for their biological activity. These data clearly suggest that the detected binding sites correspond to the physiological receptors involved in the glycogenolytic action of angiotensin on rat liver.
通过使用具有生物活性的[(3)H]血管紧张素（比放射性为14Ci/mmol）来测定大鼠肝细胞和纯化肝细胞膜上特异性血管紧张素结合情况。血管紧张素与肝细胞结合的动力学参数为：K(+1)（结合速率常数），100μmol⁻¹·min⁻¹；K(-1)（解离速率常数），2min⁻¹；K(d)（解离常数），30nmol；最大结合容量，0.42pmol/10⁶个细胞或260000个位点/细胞。血管紧张素与膜的结合受到GTP（0.1mmol）和NaCl（100mmol）的显著影响；这些调节化合物极大地提高了结合和解离速率以及解离程度。在存在GTP + NaCl的情况下，K(d)为10nmol，在不存在它们的情况下为1.5nmol；无论有无GTP + NaCl，最大结合容量均为0.70pmol/mg蛋白质。通过[(3)H]血管紧张素与肝细胞和膜结合的竞争实验推断出11种血管紧张素结构类似物的相对亲和力（在后一种情况下，不包括GTP + NaCl，以研究受体的高亲和力状态）。这些与它们的生物活性（肝细胞中糖原磷酸化酶的激活）高度相关。与膜的结合发生在与生物效应相同的浓度范围内。另一方面，激动剂与肝细胞的结合所需浓度比其生物活性所需浓度高25倍，这一观察结果提示存在大量备用受体。这些数据清楚地表明，检测到的结合位点对应于血管紧张素对大鼠肝脏糖原分解作用中涉及的生理受体。
