PMID: 9173884Apr 1, 1997Paper

Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets

The Biochemical Journal
K M TangR J Haslam

Abstract

We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEDelta1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEDelta2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEDelta1Deltai). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [32P]cGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEDelta1 (182+/-12 nM and 153+/-12 nM respectively) and cGI-PDEDelta2 (131+/-17 nM and 99+/-1 nM respectively) were significa...Continue Reading

Citations

Jun 9, 2016·Nature Chemical Biology·Lakshmi KrishnamoorthyChristopher J Chang
Jan 3, 2018·Pflügers Archiv : European journal of physiology·Sarah DünnesAndreas Friebe
Apr 15, 2000·The Journal of Biological Chemistry·Y KenanV C Manganiello
Jan 24, 2019·Neurogastroenterology and Motility : the Official Journal of the European Gastrointestinal Motility Society·Jing AnShi Liu
Dec 19, 2007·Asian Journal of Andrology·F DimitriadisN Sofikitis
May 1, 2021·Journal of Personalized Medicine·Cristina Membrive JiménezAlberto Jiménez Morales

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