Biochemical characterization of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat heart left ventricle

In the present study we investigate the bio- chemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5 0 -thymidine monophosphate (p-Nph-5 0 -TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation depen- dence and the KM value corresponded to 91.42 ± 13.97 lM and the maximal velocity (Vmax) value calculated was 63.79 ± 3.59 nmol p-nitrophenol released/min/mg of protein (mean ± SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5 0 -TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5 0 -TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had no effects on p-Nph- 5 0 -TMP hydrolysis. RT-PCR analysis of left ventricle dem- onstrated the expression of NPP2 and NPP3 enzymes, but excluded the presence of NPP1 member. By quantitative real- time PCR we identified the NPP3 as the enzyme with the highest expression in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that are involved in several cardiac pathologies.