Specific binding sites for the triiodothyronine in the plasma membrane of rat thymocytes. Correlation with biochemical responses.
Specific binding sites for the triiodothyronine in the plasma membrane of rat thymocytes. Correlation with biochemical responses.
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大鼠胸腺细胞质膜中三碘甲状腺原氨酸的特异性结合位点。
DOI：
10.1172/jci110703
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Ingbar,SH
中科院分区：
文献类型：
--
作者：
Segal,J;Ingbar,SH
作为研究大鼠胸腺细胞质膜是否含有甲状腺激素 3,5,3'-三碘甲状腺原氨酸 (T3) 的特异性、可饱和结合位点的先决条件，开发了一种从这些细胞中分离质膜部分的方法。根据电子显微镜和标记酶研究判断，该级分主要由质膜囊泡组成，不含核污染物，仅被其他亚细胞成分轻微污染。在 37°C 和 pH 7.4 下，新鲜膜制剂与 [125I]T3 的结合很快，在 5 分钟时达到最大值，然后随着时间的推移而下降，因此到 60 分钟时结合几乎为零。结合随时间的减少是由于功能性结合位点的丧失，但并不反映脱敏，因为结合活性随时间的减少与 T3 的存在或不存在无关。 Scatchard 饱和度研究分析显示存在两个结合位点，一个的表观解离常数 (Kd) 为 0.95 nM，最大结合能力为 5.3 × 1010 个位点/100 μg 蛋白，另一个结合位点的表观 Kdof 为 25 nM，结合能力为 1.4 × 1012 个位点/100 μg 蛋白。对几种甲状腺氨酸类似物抑制[125I]T3结合的能力的测量揭示了以下效力等级顺序：1-T3>1-T4>d-T3=d-T4>1-3,5-T2>rT3>d,l-甲状腺氨酸。通过从培养基中省略钙或添加β肾上腺素能拮抗剂阿普洛尔来抑制T3的结合。根据对较低亲和力结合位点的研究判断，这些操作降低了 T3 结合位点的亲和力，但没有减少其数量。甲状腺氨酸类似物抑制[125I]T3结合的相对效力通常与之前报道的在体外刺激完整大鼠胸腺细胞中糖类似物2-脱氧葡萄糖(2-DG)摄取的效力平行。此外，通过1-阿普洛尔或通过从培养基中排除钙产生的T3结合的抑制类似于先前报道的这些操作产生的关于T3诱导的2-DG摄取增强的抑制。这些发现表明，存在于大鼠胸腺细胞质膜中的 T3 结合位点充当与 T3 在这些细胞中诱导的 2-DG 摄取刺激相关的功能受体。
As a prerequisite to studies of whether the plasma membrane of the rat thymocyte contains specific, saturable binding sites for the thyroid hormone 3,5,3′-triiodothyronine (T3), a method was developed for the isolation of a plasma membrane fraction from these cells. As judged from both electron microscopic and marker enzyme studies, the fraction was composed principally of plasma membrane vesicles, was free of nuclear contaminants, and was only slightly contaminated with other subcellular components. At 37°C and pH 7.4, binding of [125I]T3by the fresh membrane preparation was rapid, reaching a maximum at 5 min and then declining with time, so that by 60 min binding was virtually nil. Decreased binding with time was due to a loss of functional binding sites, but did not reflect desensitization, since the decrease in binding activity with time was independent of the presence or absence of T3. Scatchard analysis of saturation studies revealed the presence of two binding sites, one with an apparent dissociation constant (Kd) of 0.95 nM and a maximum capacity of 5.3 × 1010sites/100 μg protein, and the other with an apparentKdof 25 nM and a binding capacity of 1.4 × 1012sites/100 μg protein. Measurement of the ability of several thyronine analogues to inhibit the binding of [125I]T3revealed the following rank order of potency:l-T3>l-T4>d-T3=d-T4>l-3,5-T2> rT3>d,l-thyronine. Binding of T3was inhibited by the omission of calcium from the medium or by the addition of the beta adrenergic antagonist alprenolol. As judged from studies of the lower affinity binding site, these manipulations decreased the affinity, but not the number, of binding sites for T3. The relative potencies of thyronine analogues to inhibit the binding of [125I]T3were generally parallel to their previously reported potencies in stimulating the uptake of the sugar analogue 2-deoxy-glucose (2-DG) in intact rat thymocytes in vitro. Further, the inhibition of T3-binding produced byl-alprenolol or by excluding calcium from the medium resembled the previously reported inhibition that these manipulations produce with respect to T3-induced enhancement of 2-DG uptake. These findings suggest that the binding sites for T3present in the plasma membrane of rat thymocytes act as functional receptors linked to the stimulation of 2-DG uptake that T3induces in these cells.