cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.
cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.
复制标题
cAMP 介导的低 Km cAMP 磷酸二酯酶的磷酸化显着刺激其催化活性。
DOI：
10.1073/pnas.85.23.9071
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发表时间：
1988
影响因子：
11.1
通讯作者：
Colman,RW
中科院分区：
文献类型：
--
作者：
Grant,PG;Mannarino,AF;Colman,RW
用腺苷酸环化酶激动剂福斯克林（100微米）处理完整的人血小板导致冻融裂解物中cAMP磷酸二酯酶活性增加。当用蓝葡聚糖- sepharose色谱法从这些裂解物中分离出低km（高亲和力）、cgmp抑制的cAMP磷酸二酯酶时，该酶的比活性比类似处理的对照血小板平均提高了11倍。在用福斯克林治疗之前，用蛋白激酶抑制剂H-8孵育血小板，可以抑制低km、cgmp抑制的cAMP磷酸二酯酶活性的增加，这表明cAMP磷酸二酯酶活性的刺激涉及cAMP依赖性磷酸化。将32Pi预标记的完整血小板用forskolin处理，并通过蓝色葡聚糖- sepharose色谱分离低km、cgmp抑制的磷酸二酯酶，磷酸化了110,000 kDa的蛋白。通过对纯化的磷酸二酯酶进行单特异性抗血清，电泳转移印迹（Western blot）和免疫沉淀分析表明该蛋白是低km， cgmp抑制的cAMP磷酸二酯酶。稳定的前列环素类似物伊洛前列素也刺激低km cAMP磷酸二酯酶活性约2倍，并引起酶磷酸化。这些结果表明，低km、cgmp抑制的磷酸二酯酶的磷酸化可能是血小板中该酶的重要调节机制。
Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.