McAllister-Lucas L M, Sonnenburg W K, Kadlecek A, Seger D, Trong H L, Colbran J L, Thomas M K, Walsh K A, Francis S H, Corbin J D
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
J Biol Chem. 1993 Oct 25;268(30):22863-73.
Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.
采用聚合酶链反应（PCR）方法和cDNA文库筛选技术，从牛肺中分离出一个编码cGMP结合型、cGMP特异性磷酸二酯酶（cGB-PDE）的cDNA克隆。基于cGB-PDE肽序列的简并寡核苷酸用作引物，以牛肺cDNA为模板进行PCR反应。回收了一个824碱基对的PCR产物，并用作探针筛选牛肺cDNA文库。分离出一个编码全长cGB-PDE的4.5千碱基对cDNA克隆。该cDNA的开放阅读框预测为一个875个氨基酸（AA）、99525道尔顿的多肽。通过Northern分析，cGB-PDE cDNA与单一的6.9千碱基肺mRNA杂交。通过将推导的AA序列与从cGB-PDE获得的几个肽序列进行比较，验证了cGB-PDE cDNA的身份。用cGB-PDE cDNA转染的COS-7细胞过表达了肺cGB-PDE特有的cGMP结合和cGMP-PDE活性。cGB-PDE的序列包含一个与所有哺乳动物磷酸二酯酶中保守的假定催化区域同源的片段（氨基酸578-812）和一个与cGMP刺激的磷酸二酯酶及光感受器磷酸二酯酶的假定cGMP结合区域同源的片段（氨基酸142-526）。正如这些磷酸二酯酶中也提到的，cGB-PDE的假定cGMP结合区域内包含两个内部同源重复序列。cGB-PDE的氨基末端142个残基与其他磷酸二酯酶没有显著同源性，并且包含被cGMP依赖性蛋白激酶磷酸化的丝氨酸（氨基酸92）。
