CHARACTERIZATION OF SP-5,6-DICHLORO-1-BETA-D-RIBOFURANOSYL-BENZIMIDAZOLE-3',5'-MONOPHOSPHOROTHIOATE (SP-5,6-DCL-CBIMPS) AS A POTENT AND SPECIFIC ACTIVATOR OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE IN CELL-EXTRACTS AND INTACT-CELLS
CHARACTERIZATION OF SP-5,6-DICHLORO-1-BETA-D-RIBOFURANOSYL-BENZIMIDAZOLE-3',5'-MONOPHOSPHOROTHIOATE (SP-5,6-DCL-CBIMPS) AS A POTENT AND SPECIFIC ACTIVATOR OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE IN CELL-EXTRACTS AND INTACT-CELLS
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DOI：
10.1042/bj2790521
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发表时间：
1991-10-15
影响因子：
4.1
通讯作者：
WALTER, U
中科院分区：
文献类型：
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作者：
SANDBERG, M;BUTT, E;WALTER, U
比较了新设计的环腺苷酸（cAMP）类似物Sp-5，6-二氯-1-β-D-呋喃核糖基苯并咪唑-3 '，5'-单硫代磷酸酯（Sp-5，6-DCl-cBiMPS）和8-（对氯苯硫基）-cAMP（8-pCPT-cAMP）的化学和生物学性质，以评估它们作为完整细胞中cAMP依赖性蛋白激酶（cAMP-PK）激活剂的潜力。SP-5，6-DCl-cBiMPS被证明是纯化的cAMP-PK和血小板膜中的cAMP-PK的有效和特异性激活剂，而8-pCPT-cAMP被证明是作为纯化的酶和血小板膜中的cAMP-PK和环GMP依赖性蛋白激酶（cGMP-PK）的有效激活剂。SP-5，6-DCl-cBiMPS不被三种类型的环核苷酸磷酸二酯酶显著水解，而8-pCPT-cAMP（和8-溴-cAMP）被Ca 2 +/钙调素依赖性磷酸二酯酶和cGMP抑制性磷酸二酯酶水解到显著程度。Sp-5，6-DCl-cBiMPS的表观亲脂性（潜在细胞膜渗透性的量度）高于8-pCPT-cAMP。将Sp-5，6-DCl-cBiMPS细胞外应用于完整的人血小板再现了前列腺素E1（一种血小板活化的cAMP增加抑制剂）诱导的蛋白磷酸化模式。在完整血小板中，Sp-5，6-DCl-cBiMPS在诱导46/50 kDa血管舒张剂刺激的磷蛋白（VASP）（血小板中cAMP-PK的主要底物）的定量磷酸化方面也比8-pCPT-cAMP更有效。正如对前列腺素E1所观察到的那样，用Sp-5，6-DCl-cBiMPS预处理人血小板可以防止凝血酶诱导的聚集。结果表明，Sp-5，6-DCl-cBiMPS是细胞提取物和完整细胞中cAMP-PK的一种非常有效且特异性的激活剂，在这方面，上级用于完整细胞研究的任何其他cAMP类似物。与8-pCPT-cAMP相比，Sp-5，6-DCl-cBiMPS可用于区分cAMP-PK和cGMP-PK介导的信号转导途径。
A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6-DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.