Terman B I, Riek R P, Grodski A, Hess H J, Graham R M
Cardiac Unit, Massachusetts General Hospital, Boston.
Mol Pharmacol. 1990 Apr;37(4):526-34.
Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.
大鼠肝脏和脑膜α1-肾上腺素能受体通过连续色谱步骤进行纯化，纯化倍数超过500倍，所使用的亲和基质包括肝素琼脂糖、通过将α1选择性拮抗剂哌唑嗪的新型衍生物偶联到Affigel-102上构建的亲和基质以及麦胚凝集素琼脂糖。利用光亲和标记、蛋白质纯化和DNA克隆技术，获得了几条证据，证明大脑中存在一种α1-肾上腺素能受体亚型，其结构与先前在肝脏和其他组织中所鉴定的不同。α1选择性配体氯乙可乐定（CEC）（一种烷基化剂）可使大鼠肝脏部分纯化制剂中100%的[3H]哌唑嗪结合位点不可逆地失活。在相同条件下，只有50%的脑受体被不可逆地失活。通过计算机模拟α拮抗剂WB4101和酚妥拉明与[3H]哌唑嗪竞争结合大鼠肝脏部分纯化制剂所获得的数据，假设两种配体都存在一类低亲和力位点时拟合效果最佳。然而，对部分纯化的脑制剂进行分析表明，这些拮抗剂存在两个具有不同亲和力的结合位点。此外，用CEC对脑受体进行预先烷基化会导致低亲和力的酚妥拉明和WB4101结合位点丧失。脑中对CEC不敏感的位点对酚妥拉明和WB4101具有高亲和力，对[125I]叠氮哌唑嗪的光亲和标记具有抗性。这并不是由于对CEC不敏感的位点对光亲和标记的亲和力明显较低，因为用[127I]叠氮哌唑嗪进行竞争研究表明，在部分纯化的脑样品中存在一类高亲和力位点。对未用CEC处理的部分纯化肝脏和脑样品进行光亲和标记，结果特异性标记出一条分子量为80,000的单一蛋白质。对于先前用CEC孵育过的部分纯化脑样品，未观察到特异性标记的蛋白质。用N-糖苷酶处理光亲和标记的肝脏和脑受体以切割N-连接寡糖，会产生一条分子量为55,000的单一蛋白质。综上所述，这些数据为大鼠肝脏中存在单一受体亚型（α1b）以及大鼠脑中存在两种亚型（α1a和α1b）提供了证据。此外，α1a亚型对CEC不敏感以及对α1b选择性光亲和探针的共价标记具有抗性，这表明两种受体亚型的一级结构不同，使得α1b亚型中包含CEC和光亲和标记的一个或多个氨基酸在α1a亚型中不存在。
