Morris D I, Robbins J D, Ruoho A E, Sutkowski E M, Seamon K B
Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, Maryland 20892.
J Biol Chem. 1991 Jul 15;266(20):13377-84.
Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.
合成了两种光标记物，即N-(3-(4-叠氮基-3-¹²⁵I-苯基)-丙酰胺)-6-氨乙基氨甲酰基福司可林(¹²⁵I-6-AIPP-Fsk)和N-(3-(4-叠氮基-3-¹²⁵I-苯基)丙酰胺)-7-氨乙基氨甲酰基-7-去乙酰基福司可林(¹²⁵I-7-AIPP-Fsk)，其比活度为2200 Ci/mmol，并用于标记腺苷酸环化酶和葡萄糖转运体。通过它们对[³H]福司可林与牛脑膜结合的抑制作用来测定光标记物对腺苷酸环化酶的亲和力。6-AIPP-Fsk和7-AIPP-Fsk抑制[³H]福司可林结合的IC50值分别为15 nM和200 nM。¹²⁵I-6-AIPP-Fsk在对照和GTPγS预激活的牛脑膜中标记了一种115-kDa的蛋白质。这种标记被福司可林抑制，但不被1,9-二脱氧福司可林或细胞松弛素B抑制。¹²⁵I-6-AIPP-Fsk对部分纯化的腺苷酸环化酶的标记被福司可林抑制，但不被1,9-二脱氧福司可林抑制。在对照和GTPγS预激活的脑膜中，¹²⁵I-7-AIPP-Fsk特异性地标记了一种45-kDa的蛋白质，而不是115-kDa的蛋白质。这种标记被福司可林、1,9-二脱氧福司可林、细胞松弛素B和D-葡萄糖抑制，但不被细胞松弛素E或L-葡萄糖抑制。用人红细胞膜与¹²⁵I-6-AIPP-Fsk和¹²⁵I-7-AIPP-Fsk进行光解。¹²⁵I-7-AIPP-Fsk强烈标记了一条宽的45-70-kDa条带，而¹²⁵I-6-AIPP-Fsk则没有。福司可林、7-溴乙酰基-7-去乙酰基福司可林、1,9-二脱氧福司可林、细胞松弛素B和D-葡萄糖，但不包括细胞松弛素E或L-葡萄糖，抑制了¹²⁵I-7-AIPP-Fsk对45-70-kDa条带的标记。¹²⁵I-6-AIPP-Fsk和¹²⁵I-7-AIPP-Fsk分别是对腺苷酸环化酶和葡萄糖转运体具有特异性的高亲和力光标记物。
