Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents.
Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents.
复制标题
主要血小板低 Km cAMP 磷酸二酯酶的免疫学鉴定：抗血栓药物的可能靶点。
DOI：
10.1073/pnas.83.17.6660
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发表时间：
1986
影响因子：
11.1
通讯作者：
Beavo,JA
中科院分区：
文献类型：
--
作者：
Macphee,CH;Harrison,SA;Beavo,JA
免疫印迹和酶活性分析，使用特定的免疫学探针，表明牛和人血小板中存在的总低KM-cAMP磷酸二酯酶活性的80%以上存在于单一的磷酸二酯酶同工酶中。在蛋白酶抑制剂存在的情况下，血小板酶的表观亚基大小为110 kDa，在免疫和结构上似乎与最近纯化的牛心同工酶没有区别。当匀浆和离心过程中没有蛋白酶抑制剂时，这种血小板磷酸二酯酶容易被顺序的蛋白分解，形成80 kDa和60 kDa的多肽。正如先前关于纯化血小板低千米cAMP磷酸二酯酶的报道所描述的那样，我们的数据表明这是一个蛋白水解性片段。此外，在我们的研究中，催化活性的40%-70%的增加与蛋白质分解有关。药理学研究证明了血小板和心脏磷酸二酯酶之间的进一步相似之处，表明这两种酶的抑制物谱相同。几种已知的磷酸二酯酶抑制剂化合物，已被发现有效地抑制血小板聚集，也抑制血小板低KM-cAMP磷酸二酯酶，其效力与其抗血栓作用非常相似。西洛胺、Ro15-2041、米力农、罂粟碱、异丁基甲基黄嘌呤和茶碱对110 kDa血小板酶有抑制作用，IC50值分别为0.04、0.13、0.46、1.4、2.6和110微米。
Immunoblot and enzyme-activity analyses, using specific immunological probes, indicated that more than 80% of the total low-Km cAMP phosphodiesterase activity present in bovine and human platelets resided in a single phosphodiesterase isozyme. In the presence of protease inhibitors, the platelet enzyme has an apparent subunit size of 110 kDa and appears immunologically and structurally indistinguishable from a recently purified bovine heart isozyme. When protease inhibitors were absent during homogenization and centrifugation, this platelet phosphodiesterase was susceptible to sequential proteolysis forming 80-kDa and 60-kDa peptides. As a previous report on the purification of the platelet low-Km cAMP phosphodiesterase described a 61-kDa protein, our data would suggest that this was a proteolytic fragment. Moreover, in our study a 40-70% increase in catalytic activity was associated with proteolysis. Further similarities between the platelet and heart phosphodiesterases were demonstrated by pharmacological studies that showed identical inhibitor profiles for both enzymes. Several known phosphodiesterase inhibitor compounds that have been found useful in inhibiting platelet aggregation also inhibited the platelet low-Km cAMP phosphodiesterase with potencies very similar to their antithrombotic effects. Cilostamide, Ro 15-2041, milrinone, papaverine, isobutylmethylxanthine, and theophylline inhibited the 110-kDa platelet enzyme with IC50 values of 0.04, 0.13, 0.46, 1.4, 2.6, and 110 microM, respectively.