Hauger R L, Hulihan-Giblin B, Skolnick P, Paul S M
Life Sci. 1984 Feb 20;34(8):771-82. doi: 10.1016/0024-3205(84)90385-0.
Recent studies in our laboratory have demonstrated the presence of specific binding sites for [3H] (+)-amphetamine in crude membrane preparations derived from rat brain. In this report we have further characterized the specific binding of [3H] (+)-amphetamine in various subcellular fractions of rat brain and demonstrate a greater than five-fold enrichment in the crude synaptosomal (P2) fraction compared to a crude membrane preparation. Specific [3H] (+)-amphetamine binding in crude synaptosomal membranes is saturable and stereospecific with an apparent dissociation constant, Kd, of 2.8 +/- 0.5 microM and an estimated maximum number of binding sites, Bmax, of 60.4 +/- 8.4 pmoles/mg protein derived by Scatchard or Klotz analysis of binding data using filtration assays. Centrifugation assays yield a similar Kd though the apparent Bmax is higher. In addition specific [3H] (+)-amphetamine binding is: rapidly reversible, temperature sensitive, labile to preincubation in Tris buffer, inhibited by sodium ions and unevenly distributed in various brain regions. Specific [3H] (+)-amphetamine binding sites are found almost exclusively in the rat central nervous system (the brainstem, hypothalamus, and striatum exhibiting relatively high levels of binding), whereas peripheral tissues such as liver, kidney and heart have very low to undetectable levels of specific binding.
我们实验室最近的研究表明，在源自大鼠脑的粗制膜制剂中存在[3H]（+）-苯丙胺的特异性结合位点。在本报告中，我们进一步对大鼠脑不同亚细胞组分中[3H]（+）-苯丙胺的特异性结合进行了表征，并证明与粗制膜制剂相比，粗制突触体（P2）组分中的富集度超过五倍。粗制突触体膜中特异性[3H]（+）-苯丙胺结合是可饱和的且具有立体特异性，其表观解离常数Kd为2.8±0.5微摩尔，通过使用过滤测定法对结合数据进行Scatchard或Klotz分析得出的估计最大结合位点数Bmax为60.4±8.4皮摩尔/毫克蛋白质。离心测定法得出的Kd相似，尽管表观Bmax较高。此外，特异性[3H]（+）-苯丙胺结合具有以下特点：快速可逆、对温度敏感、在Tris缓冲液中预孵育不稳定、受钠离子抑制且在不同脑区分布不均。特异性[3H]（+）-苯丙胺结合位点几乎只存在于大鼠中枢神经系统中（脑干、下丘脑和纹状体表现出相对较高的结合水平），而肝脏、肾脏和心脏等外周组织的特异性结合水平非常低或无法检测到。
