Solubilization and substrate specificity of membrane-bound nucleotide phosphodiesterase-pyrophosphohydrolase activities from Dictyostelium discoideum.
Solubilization and substrate specificity of membrane-bound nucleotide phosphodiesterase-pyrophosphohydrolase activities from Dictyostelium discoideum.
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盘基网柄菌膜结合核苷酸磷酸二酯酶-焦磷酸水解酶活性的溶解和底物特异性。
DOI：
10.1016/s0021-9258(18)32229-4
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
J. Jahngen
中科院分区：
文献类型：
--
作者：
E. Rossomando;J. Jahngen
描述了从盘基网柄菌无细胞裂解物制备的膜富集组分中分离出裂解a-6-磷酸酸酐键和磷酸二酯键的部分纯化活性。膜的洗涤剂提取物使这两种活性溶剂化，并用DE-52柱层析进一步分离。焦磷水解酶活性用类似物5‘-(B，~-亚甲基)三磷酸(AMPPCP)和腺基5’-酰亚胺二磷酸(AMPPNP)测定，并用三磷酸腺苷(ATP)测定。以三磷酸腺苷为底物的动力学研究表明，蛋白质的K值为55 PM，V值为32nmoI/min/mg蛋白质。以AMPPNP、AMPPCP或福尔马菌素5“三磷酸(FoATP)为底物，三种化合物均可获得约100 PM和23-25nmoI/min/mg蛋白质的值。以荧光类似物FoATP为底物，研究了ATP、AMPPCP和AMPPNP对反应的影响，发现它们是竞争性抑制剂，其表观KI值分别为100、100和220 PM。ADP也是一种竞争性抑制剂，其表观KI值为280
The partial purification of activities that cleave an a-6-phosphoanhydride bond and a phosphodiester bond isolated from a membrane-enriched fraction prepared from cell-free lysates of Dictyostelium discoideum is described. Detergent extraction of the membranes sol-ubilized both activities which were separated further by DE-52 column chromatography. The pyrophosphohydrolase activity was assayed with the analogs adenosine 5 ‘-(B,~-methyIene) triphosphate (AMPPCP) and adenyl 5’-yl imidodiphosphate (AMPPNP) as well as with ATP. Kinetic studies with ATP as substrate revealed a K,,, of 55 PM and a V,, of 32 nmoI/min/mg of protein. With AMPPNP, AMPPCP, or formycin 5” triphosphate (FoATP) as substrate, K,,, and V,,, values of approximately 100 PM and 23-25 nmoI/min/mg of protein were obtained for all three compounds. With the fluorescent analog FoATP as the substrate, the effects of ATP, AMPPCP, and AMPPNP on the reaction were studied and found to be competitive inhibitors with apparent Ki values of 100, 100, and 220 PM, respectively. ADP was also a competitive inhibitor with an apparent Ki value of 280