Continuous fluorimetric assay of nucleotide pyrophosphatase. Kinetics, inhibitors, and extension to dinucleoside oligophosphatases

Abstract

A simple and convenient procedure is described for the continuous fluorimetric assay of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) activity. It is based on the multifold increase in fluorescence emission intensity accompanying hydrolysis of a new fluorogenic substrate, ϵAp 2ϵA (where ϵ = 1, N 6-etheno), or of commercially available ϵNAD + and FAD, using potato nucleotide pyrophosphatase. The procedure is applicable to enzyme activity in tissue extracts, as well as to kinetic studies and screening of potential inhibitors. K m and V max/ K m for the substrates, and K i values for various compounds, were evaluated. The most effective inhibitor of ϵAp 2ϵA hydrolysis was the alternative substrate 8-bromo-NAD + ( K i = 7 μM). The method is also useful for continuous assay of snake venom phosphodiesterase I-5′-exo-nuclease (EC 3.1.4.1). The preparation of ϵAp 2ϵA is described, and spectral properties of the fluorogenic substrates listed. Both ϵAp 3ϵA and ϵAp 4ϵA were also shown to be fluorogenic substrates for nucleotide pyrophosphatase and phosphodiesterase I, and may be used for continuous fluorimetric assay of specific dinucleoside oligophosphatases.