RESUMO
Recombinant expression in Escherichia coli of human cyclic nucleotide phosphodiesterase 4B2 (hPDE4B2) fused to maltose-binding-protein (MBP-hPDE4B2) was investigated. hPDE4B2 DNA amplified via nested RT-PCR with total RNAs from U937 cells was ligated with pMAL-p2x. After induction at 18 degrees C for 16 h, soluble MBP-hPDE4B2 was produced in E. coli. MBP-hPDE4B2 after amylose-resin chromatography showed 35% homogeneity, and its Michaelis-Menten constant was 10+/-2 microM (n=3). Rolipram had a dissociation constant of 9+/-2 nM (n=2), and zinc ion was a potent inhibitor. Hence, MBP-hPDE4B2 was expressed in E. coli as a soluble active protein.
Assuntos
Proteínas de Transporte/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/biossíntese , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Escherichia coli/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Animais , Extratos Celulares , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/isolamento & purificação , Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isopropiltiogalactosídeo/farmacologia , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
A spectrometric method was investigated to measure the activities of recombinant human cyclic nucleotide phosphodiesterase 4 (PDE4), 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 (K(m)) of (12.0+/-2.1)microM (n=3) for AMP at pH 7.4, and was resistant to EDTA below 0.20mM. By the coupled end-point assay at 30.0UL(-1) CIAP with reaction durations within 30min, the rates to release phosphate in PDE4 reaction mixtures containing 10.0mM MgCl(2) and 0.10mM EDTA linearly responded to the amounts of PDE4 over wide ranges. Meanwhile, K(m) of PDE4 was (8.8+/-0.2)microM (n=2), zinc ion inhibited PDE4 and rolipram had the inhibition constant about 10nM. These results supported that by the coupled end-point assay, this method was promising to screen of PDE inhibitors that had no interference with the MLG assay of phosphate.
