title: Microplate-based method to screen inhibitors of isozymes of cyclic nucleotide phosphodiesterase fused to SUMO.
abstract: The feasibility for microplate-based screening of inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE) was tested via the coupled action of a phosphatase on adenosine-5'-monophosphate and an improved malachite green assay of phosphate. Human full-length PDE4B2 and truncated mutant (152-528aa) were expressed in Escherichia coli via fusion to SUMO, which after purification through Ni-NTA column exhibited specific activities >0.017 U mg(-1). In the presence of proteins <30 mg L(-1), absorbance for 10 µΜ phosphate was measurable; a PDE isozyme of specific activity over 0.008 U mg(-1) after reaction for 20 min thus suited for microplate-based screening of inhibitors. By using Biotek ELX 800 microplate reader, affinities of two forms of PEDE4B2 for cAMP, rolipram and papaverine varied over three magnitudes and were consistent with those by routine assay, respectively. Hence, the proposed method was promising for high-throughput-screening of inhibitors of phosphate-releasing enzymes bearing specific activities over 0.008 U mg(-1).
authors: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University , Chongqing 400016 , China.
authors: Chunyan
authors: C
authors: Chen
authors: Miaomiao
authors: M
authors: Liu
authors: Jing
authors: J
authors: Wu
authors: Xiaolan
authors: X
authors: Yang
authors: Xiaolei
authors: X
authors: Hu
authors: Jun
authors: J
authors: Pu
authors: Gaobo
authors: G
authors: Long
authors: Yanling
authors: Y
authors: Xie
authors: Hairong
authors: H
authors: Jiang
authors: Yonghua
authors: Y
authors: Yuan
authors: Fei
authors: F
authors: Liao
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D000242:Cyclic AMP / Q000737:chemistry; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000737:chemistry* / Q000235:genetics; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D010208:Papaverine / Q000737:chemistry*; D010710:Phosphates / Q000032:analysis*; D058988:Phosphodiesterase 4 Inhibitors / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D020889:Rolipram / Q000737:chemistry*; D012394:Rosaniline Dyes / Q000737:chemistry; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D007527:Isoenzymes; D010710:Phosphates; D058988:Phosphodiesterase 4 Inhibitors; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; D025841:Small Ubiquitin-Related Modifier Proteins; C005095:malachite green; D000249:Adenosine Monophosphate; D010208:Papaverine; D000242:Cyclic AMP; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517360:PDE4B protein, human; D020889:Rolipram
keywords: An improved malachite green assay o phosphate; cyclic nucleotide phosphodiesterase; high-throughput-screening; inhibitor