Screening Of Cyclic Nucleotide Phosphodiesterase 4 Inhibitors Via Malachite Green Assay Of Inorganic Phosphate Released By Calf Intestinal Alkaline Phosphatase

Recombinant expression in E. coli of human cyclic nucleotide phosphodiesterase 4B2 (hPDE4B2) fused to maltose-binding-protein (MBP-hPDE4B2) was investigated. hPDE4B2 DNA was ligated with pMAL-p2x. After induction at 18 0C for 16 h, soluble MBP-hPDE4B2 was produced in E. coli. One-step amylose-resin chromatography purified MBP-hPDE4B2 to about 35% homogeneity. MBP-hPDE4B2 had Michaelis-Menten constant of (9.6±1.8)μ?M (n = 3) and was sensitive to zinc ion and rolipram. This MBP-hPDE4B2 had the specific activity about 0.1 U.mg-1, and could be used for screening of PDE4 inhibitors.A spectrometric method was investigated to measure the activities of recombinant MBP-hPDE4B2, based on the use of malachite green (MLG) to quantify phosphate released from adenosine-5'-monophosphate (AMP) by the action of calf intestinal alkaline phosphatase (CIAP). Glycerol at 2% stabilized the complex between MLG and phosphomolybdate, whose absorbance at 630nm was proportional to phosphate concentrations with resistance to common substances in PDE4 reaction mixtures except papaverine. CIAP had the Michaelis–Menten constant (Km) of (12.0±2.1)μ?M (n = 3) for AMP at pH 7.4, and was resistant to EDTA below 0.20mM. By the coupled end-point assay at 30.0 U·L?1 CIAP and reaction within 30 min, lagging time for steady-state reaction was about 1.0 min, amounts of phosphate released in reaction mixtures linearly responded to the amounts of PDE4 over wide ranges. So, this method was promising to screen of PDE inhibitors having no interference with the MLG assay of phosphate.By this MLG assay of phosphate coupled to CIAP action, Km of MBP-hPDE4B2 and other PDEs was successfully determined. On MBP-hPDE4B2, rolipram had the inhibition constant was 11 nM. And with other sources of PDEs, inhibitors without interferences with MLG assay of phosphate were screened. With microplate system, target enzyme should be as small as possible to enable analyses of phosphate without removal of denatured proteins. With CIAP of much higher specific activity and benzidine as a model, this MLG assay of phosphate was effective without removal of proteins in microplate reaction system. Therefore, this MLG assay of phosphate coupled to CIAP was promising to screen of PDE inhibitors as long as target enzyme of high specific activity was available.