Stiles G L, Benovic J L, Caron M G, Lefkowitz R J
J Biol Chem. 1984 Jul 10;259(13):8655-63.
Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
哺乳动物β-肾上腺素能受体结合肽可以通过用光亲和试剂对-叠氮基间-[¹²⁵I]碘苄咔唑醇进行共价标记，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影来可视化。受体肽迁移为Mr约等于62,000的宽带。在本研究中，我们通过使用特异性外切糖苷酶和内切糖苷酶以及凝集素亲和色谱法研究了哺乳动物β受体的碳水化合物组成。用外切糖苷酶神经氨酸酶和α-甘露糖苷酶处理来自仓鼠肺或大鼠红细胞的对-叠氮基间-[¹²⁵I]碘苄咔唑醇标记的β₂-肾上腺素能受体，为β₂-肾上腺素能受体上同时存在高甘露糖型和复合型碳水化合物链提供了证据。这些酶顺序处理的效果不具有加和性，提示存在含有复合型或高甘露糖型链的不同群体的β-肾上腺素能受体。用内切糖苷酶F对受体进行去糖基化处理后，两个系统均产生一条Mr = 49,000的单一标记多肽。通过对溶解的对-叠氮基间-[¹²⁵I]碘苄咔唑醇标记的受体进行凝集素亲和色谱法，也可以分离出相同的两个β受体群体（高甘露糖型或复合型链）。含高甘露糖的受体可以被刀豆球蛋白A-琼脂糖吸附并特异性洗脱。含复合型碳水化合物的受体可以被麦胚凝集素-琼脂糖吸附并洗脱。综上所述，这些数据表明哺乳动物β-肾上腺素能受体同时含有复合型和高甘露糖型碳水化合物链，并且这些链的微异质性可能解释了在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上通常获得的宽带模式。
