Meacci E, Taira M, Moos M, Smith C J, Movsesian M A, Degerman E, Belfrage P, Manganiello V
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1992 May 1;89(9):3721-5. doi: 10.1073/pnas.89.9.3721.
We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in lambda Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxyl-terminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.
我们从λZap II载体中的人心脏cDNA文库中克隆了心肌cGMP抑制性cAMP磷酸二酯酶（cGI PDE）的cDNA。cDNA克隆n.13.2（7.7 kb）的开放阅读框[3.5千碱基（kb）]编码一个125 kDa的蛋白质。在人心室总RNA的Northern印迹中，一个单一的mRNA种类（8.3 kb）与克隆n.13.2 cDNA的一个4 kb EcoRI限制性片段（包含整个开放阅读框）杂交。cGI PDE推导氨基酸序列的羧基末端区域包含在哺乳动物PDE家族中保守的假定催化结构域。一个编码截短的、含有保守结构域的54 kDa cGI PDE的部分cDNA克隆n.2在大肠杆菌中表达为具有催化活性的融合蛋白。cAMP水解活性受到cGMP和OPC 3911的抑制，但不受咯利普兰的抑制。因此，本报告提供了直接证据，证明保守结构域包含cGI PDE的催化核心。
