Harrison S A, Reifsnyder D H, Gallis B, Cadd G G, Beavo J A
Mol Pharmacol. 1986 May;29(5):506-14.
We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs.
我们已从牛心肌中鉴定并高度纯化出一种“低 Km”的环磷酸腺苷（cAMP）磷酸二酯酶。这种磷酸二酯酶受到低浓度环磷酸鸟苷（cGMP）的抑制，因此被暂时命名为 cGMP 抑制性磷酸二酯酶。在比活性提高 16000 倍后，高度纯化的酶比活性为 6 μmol/分钟 - 毫克，并含有三种主要的多肽。初步数据表明，所有这些多肽都是通过蛋白水解从单一共同前体衍生而来。我们使用这种酶制剂制备了针对“低 Km”磷酸二酯酶的多克隆抗血清和单克隆抗体。免疫吸附和免疫印迹分析使我们能够鉴定和分离出几种磷酸二酯酶的分子量种类，包括一种比之前报道的任何纯化的低 Km 磷酸二酯酶都更大的形式。这种大形式的酶亚基分子量约为 110000，是心肌新鲜提取物中唯一可见的一种。在磷酸二酯酶 - 抗体复合物中恢复了完全的催化活性，通过免疫沉淀制备的酶对 cAMP 水解和 cGMP 抑制表现出米氏动力学。cAMP 水解的 Km 为 0.15 μM，cGMP 对 cAMP 水解抑制的 Ki 为 0.06 μM。这种免疫沉淀方法还使我们能够确定该酶被 cAMP 依赖性蛋白激酶磷酸化在丝氨酸残基上，并且低 Km、cGMP 抑制性磷酸二酯酶被几种新型强心剂选择性抑制。米力农、氨力农和非诺昔酮是这种同工酶的高度选择性抑制剂这些抑制剂的相对亲和力与它们作为正性肌力药物的效力顺序一致。这些研究表明，cGMP 抑制性磷酸二酯酶是几种新型强心药物的受体。
