The measurement of cyclic nucleotide phosphodiesterase 4 activities via the quantification of inorganic phosphate with malachite green.
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.
Zhu
Sha
S
Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, College of Pharmaceutical Sciences, Chongqing Medical University, Chongqing 400016, China.
Gan
Zhiyong
Z
Li
Zhirong
Z
Liu
Yin
Y
Yang
Xiaolan
X
Deng
Ping
P
Xie
Yanlin
Y
Yu
Mingan
M
Liao
Hong
H
Zhao
Yunsheng
Y
Zhao
Lina
L
Liao
Fei
F
D000249:Adenosine Monophosphate / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004396:Coloring Agents / Q000737:chemistry*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008982:Molybdenum / Q000737:chemistry; D010710:Phosphates / Q000032:analysis*; D058988:Phosphodiesterase 4 Inhibitors; D010756:Phosphoric Acids / Q000737:chemistry; D012394:Rosaniline Dyes / Q000032:analysis / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods*
D004396:Coloring Agents; D010710:Phosphates; D058988:Phosphodiesterase 4 Inhibitors; D010756:Phosphoric Acids; D012394:Rosaniline Dyes; C005095:malachite green; D000249:Adenosine Monophosphate; D008982:Molybdenum; D000469:Alkaline Phosphatase; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C003125:phosphomolybdic acid