Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.
Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.
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通过亲和层析从火鸡红细胞膜中分离无腺苷酸环化酶的 β-肾上腺素能受体。
DOI：
10.1073/pnas.74.9.3710
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发表时间：
1977
影响因子：
11.1
通讯作者：
A. Strosberg
中科院分区：
文献类型：
--
作者：
G. Vauquelin;P. Geynet;J. Hanoune;A. Strosberg
腺苷酸环化酶[ATP焦磷酸裂解酶（环化）; EC 4.6.1.1]和火鸡红细胞质膜的β-肾上腺素能受体通过用NaF或鸟苷酰亚胺二磷酸和毛地黄皂苷处理以活性形式溶解。溶解的酶不再刺激的儿茶酚胺，氟化钠，或鸟嘌呤核苷酸。毛地黄皂苷提取物在alprenolol-琼脂糖衍生物上进行色谱分析。虽然大部分蛋白质和所有腺苷酸环化酶活性不受阻碍地通过柱，但受体被保留。用含有1 M NaCl的阿普洛尔溶液洗脱，使其不含酶活性;产率为25- 30%。阿普洛尔洗脱液的蛋白质含量太低，无法通过Lowry技术进行估计，并通过更灵敏的荧光法进行评估。在这些条件下，β-肾上腺素能受体在单一步骤中纯化约2000倍，保留其所有药理学性质。这些实验证实，β-肾上腺素能受体和腺苷酸环化酶是独立的实体，可以在功能基础上分离。
The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis.