Hnatowich M R, Queen G, Stein D, LaBella F S
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Can J Physiol Pharmacol. 1989 Jun;67(6):568-76. doi: 10.1139/y89-091.
We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
我们已经证实在大鼠脑匀浆中，生物活性促肾上腺皮质激素（ACTH）的酪氨酸23单碘化衍生物[125I][苯丙氨酸2，异亮氨酸4]ACTH 1 - 24存在特异性、高亲和力结合。同样，在代谢抑制和未抑制的大鼠全脑切片中，存在一个依赖于时间、蛋白质浓度和pH值的特异性“结合 - 隔离”过程。在匀浆中，结合曲线最好用双位点模型描述，并提供了以下参数：Kd1 = 0.65±0.47 nM，Bmax1 = 21±41 fmol/mg蛋白质；Kd2 = 97±48 nM，Bmax2 = 3.5±1.8 pmol/mg蛋白质。在代谢活性的脑切片中，[125I][苯丙氨酸2，异亮氨酸4]ACTH 1 - 24结合 - 隔离的浓度竞争曲线可用三个成分描述（Kd1 = 14±24 nM，Bmax1 = 50±95 fmol/mg蛋白质；Kd2 = 2.4±1.9 microM，Bmax2 = 44±49 pmol/mg蛋白质；Kd3 = 0.16±1.0 mM，Bmax3 = 5.3±54 nmol/mg蛋白质）。通过去除葡萄糖并添加100 microM哇巴因进行代谢抑制，仅消除了最低亲和力、最高容量的结合 - 隔离成分（Kd1 = 7.1±14 nM，Bmax1 = 8.7±16 fmol/mg蛋白质；Kd2 = 7.4±4.49 microM，Bmax2 = 37±27 pmol/mg蛋白质）。从代谢抑制的组织切片获得的两个结合 - 隔离参数估计值与未抑制组织获得的两个较高亲和力成分的估计值无显著差异。因此，代谢抑制仅允许显示ACTH受体结合，而不受配体：受体复合物的隔离或内化干扰。（摘要截断于250字）
