Resonant-Mie-scattering of aggregates of phosphomolybdate and papaverine for measuring activities and screening inhibitors of cyclic nucleotide phosphodiesterase isozymes

Abstract

A resonant-light-scattering (RLS) method was proposed to quantify phosphate for screening inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE). In acidified mixtures of phosphate, papaverine and molybdate, there were aggregates exhibiting micrometre sizes, no absorbance peaks over 360 nm but strong RLS peaks at 392 nm; Mie scattering thus accounted for the RLS signals. When papaverine was added before molybdate to acidified samples of phosphate, RLS signals at 392 nm were stable from 5 to 25 min since the addition of molybdate; after optimization, phosphate from 0.40 to 3.60 μM was quantifiable. This RLS method tolerated 60 mg L(-1) proteins besides common PDE inhibitors and dimethyl sulfoxide in acidified samples of phosphate; the integration of this RLS method with the coupled action of a phosphomonoesterase on PDE product was thus rational to measure PDE activities without the removal of proteins in samples. By quantifying activities of a truncated mutant of human PDE4B2 via this RLS method, Michaelis-Menten constant, inhibition constants of rolipram, papaverine and theophylline varied over three magnitudes and were consistent with those estimated by an improved malachite green assay of phosphate, respectively. Hence, this novel RLS method was promising for screening inhibitors of PDE isozymes.

Keywords: 3′,5′-cyclic adenosine monophosphate; 5′-adenosine monophosphate; 5′-guanosine monophosphate; AMP; CIAP; Cyclic nucleotide phosphodiesterase; GMP; Inhibitor; PDE; PVA; Papaverine; Phosphomolybdate; RLS; Resonant-light-scattering; Screening; cAMP; calf intestinal alkaline phosphatase; cyclic nucleotide phosphodiesterase; poly(vinyl alcohol); resonant-light-scattering.