Onali P, Strada S J, Chang L, Epstein P M, Hersh E M, Thompson W J
Cancer Res. 1985 Mar;45(3):1384-91.
Soluble, high-affinity cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterases extracted from blast cells of patients with acute myelogenous leukemia have been characterized by physical, kinetic, and immunological criteria and fractionated to a high degree of purity. Procedures used in this study were similar to those used to purify the high-affinity enzyme from dog kidney. Two forms of high-affinity enzyme were found in blast cells. Form A was similar to the known type IV phosphodiesterases, including those of normal lymphocytes and monocytes. It showed a molecular weight near 60,000, a rate of hydrolysis of cyclic AMP 7 to 10 times that of cyclic guanosine 3':5'-monophosphate (cyclic GMP), competitive inhibition by cyclic GMP for cyclic AMP hydrolysis, and identical immunoreactivity by Western transfer analysis. This enzyme form was purified to apparent homogeneity by physical criteria but showed a low maximum velocity relative to other phosphodiesterase forms. A second, different form of high-affinity phosphodiesterase (Form B) was also resolved and partially purified. By comparison with Form A, this enzyme eluted from diethylaminoethyl cellulose at slightly lower ionic strength, had a lower molecular weight, appeared specific for cyclic AMP as substrate, showed no inhibition of cyclic AMP hydrolysis by cyclic GMP, and displayed no immunological cross-reactivity to the Mr 60,000 enzyme. Neither enzyme form was activated by calmodulin or proteolysis, whereas both showed comparable inhibition by 6,7-dimethoxy-1-veratrylisoquinoline, 1-methyl-3-isobutylxanthine, and 1,3-dimethylxanthine.
从急性髓性白血病患者原始细胞中提取的可溶性、高亲和力环磷酸腺苷（cAMP）磷酸二酯酶，已通过物理、动力学和免疫学标准进行了表征，并被高度纯化。本研究中使用的方法与用于从狗肾中纯化高亲和力酶的方法相似。在原始细胞中发现了两种高亲和力酶形式。A 型与已知的 IV 型磷酸二酯酶相似，包括正常淋巴细胞和单核细胞中的那些酶。它的分子量接近 60,000，cAMP 的水解速率是环磷酸鸟苷（cGMP）的 7 至 10 倍，cGMP 对 cAMP 水解有竞争性抑制作用，并且通过 Western 印迹分析显示免疫反应性相同。这种酶形式通过物理标准纯化至表观均一性，但相对于其他磷酸二酯酶形式显示出较低的最大速度。还分离并部分纯化了第二种不同形式的高亲和力磷酸二酯酶（B 型）。与 A 型相比，这种酶在略低的离子强度下从二乙氨基乙基纤维素上洗脱，分子量较低，对 cAMP 作为底物具有特异性，不受 cGMP 对 cAMP 水解的抑制，并且对分子量为 60,000 的酶没有免疫交叉反应性。两种酶形式均未被钙调蛋白或蛋白水解激活，而两者均显示出被 6,7 - 二甲氧基 - 1 - 藜芦基异喹啉、1 - 甲基 - 3 - 异丁基黄嘌呤和 1,3 - 二甲基黄嘌呤可比的抑制作用。
