SYNTHETIC TOOLS FOR ADRENOCORTICOTROPIN RECEPTOR IDENTIFICATION
SYNTHETIC TOOLS FOR ADRENOCORTICOTROPIN RECEPTOR IDENTIFICATION
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DOI：
10.1021/bi00329a024
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
HOFMANN, K
中科院分区：
文献类型：
--
作者：
FINN, FM;STEHLE, CJ;HOFMANN, K
Biotinylated photoaffinity derivatives of ACTH are potentially useful tools for the identification of ACTH receptors. The hormone can be attached covalently to its receptor by photoactivation and the presence of biotin in the molecule facilitates isolation of the solubilized hormone-receptor complex on columns of immobilized succinoylavidin (Suc-avidin). Six photoprobes of ACTH1-24 were prepared by reacting ACTH1-24, [25-biocytin]ACTH1-25 amide and [25-dethiobiocytin]ACTH1-25 amide with either 4- or 5-azido-2-nitrophenylsulfenyl (4-NAPS and 5-NAPS, respectively) chlorides in acetic acid. The homogeneity of the photoprobes was carefully monitored by thin-layer chromatography and amino acid analyses of acid hydrolysates. The presence of underivatized starting material in the photoprobes was critically scrutinized by high-pressure liquid chromatography and was < 0.5%. Both the 4- and 5-NAPS derivatives stimulated maximal steroidogenesis (as compared with ACTH1-24) in calf adrenal cortical cells. The potencies of the 2 isomers differed significantly. The ED50 for steroidogenesis with 5-NAPS-ACTH1-24 was 100-fold greater than the standard (ACTH1-24) while that for 4-NAPS-ACTH1-24 was only .apprx. 7 times greater. Although 4-NAPS-ACTH1-24 was capable of stimulating maximal cAMP production, the 5-NAPS derivative was usually not. The level of stimulation with the 5-NAPS derivative varied considerably from cell preparation to cell preparation. ACTH1-24-induced cAMP production was inhibited by 5-NAPS-ACTH1-24 or 5-NAPS-[25-dethiobiocytin]ACTH1-25 amide. For these derivatives to be useful for isolation of covalent photoprobe-receptor complexes, it was important to ascertain that they bind to adrenal cells in the presence in Suc-avidin. [25-Biocytin]ACTH1-25 amide retained 10%, on a molar basis, of its steroidogenic activity and cAMP stimulating activity when assayed in the presence of an equimolar amount of Suc-avidin. The activity did not decrease further when the molar ratio of Suc-avidin to [25-biocytin]-ACTH1-25 amide was increased to 10:1. The findings that the 5-NAPS derivatives stimulate cAMP and steroid production and inhibit cAMP formation due to ACTH1-24 indicate that they interact with ACTH receptors. It could be expected that if irradiation of a photoaffinity-labeled hormone caused covalent attachment of the hormone to its cell surface receptor, then activation of hormone-dependent events should persist even after the cells were washed free of unattached hormone. Such was the case with stimulation of cAMP production by 5-NAPS-[25-dethiobiocytin]ACTH1-25 amide. In the absence of irradiation, the photoprobe was apparently removed from the cells by washing. Photoactivation of the hormone analog resulted in persistent activation when the cells were subsequently incubated without hormone. The 4-NAPS derivatives failed to elicit elevated cAMP production after the cells were washed. Irradiation of calf adrenal cortical cells exposed to 5-NAPS derivatives of ACTH may bring about covalent attachment of the hormone to or near the ACTH receptors.
