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

Abstract

In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence and the K ( M ) value corresponded to 91.42 +/- 13.97 microM and the maximal velocity (V ( max )) 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'-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5'-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'-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated 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.

Biochemical characterization of an ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat cardiac soluble and microsomal fractions

“…Considering the lack of effect of LPA as an inhibitor of NPP2, the effects of suramin could suggest the presence of NPP1 on both fractions; however, the involvement of NPP3 on p-Nph-5′-TMP hydrolysis may not discarded once it was not investigated. All together our results suggest that under the conditions tested, we worked with a predominant E-NPP activity both in soluble and microsomal fractions 14,20,28 . Michaelis constant (K M ) and V max calculated from the Eadie-Hofstee plot with p-Nph-5′-TMP as substrate demonstrated values that are in accordance with previous studies related to E-NPPs 26,29,32 .…”

“…These results are in accordance with the literature that demonstrated NPP1-3 as metalloenzymes 31 . In addition, with regard to E-NPPs, the pH curves demonstrated a maximal enzymatic activity at alkaline pH 20,31 . The possible participation of other enzyme activities in the substrate hydrolysis was discarded by the use of several compounds like levamisole, sodium azide and gadolinium chloride, which had no effects.…”

“…However, subcellular distribution and the exact participation of this enzyme activity on nucleotide hydrolysis are poorly understood. In a recent study, Rücker et al 20 investigated the expression of NPP1-3 in rat heart left ventricles, and observed only the NPP2 and NPP3 expression in that tissue; however, NPP1 expression have also been demonstrated in the heart by other researchers 31 . In addition to demonstrating expression, Rücker et al 20 characterized the biochemical properties of NPP activity in cardiac synaptosomes and suggested the possible involvement of these enzymes in the control of nucleotides and nucleoside levels in the sympathetic nerve ends.…”

“…In a recent study, Rücker et al 20 investigated the expression of NPP1-3 in rat heart left ventricles, and observed only the NPP2 and NPP3 expression in that tissue; however, NPP1 expression have also been demonstrated in the heart by other researchers 31 . In addition to demonstrating expression, Rücker et al 20 characterized the biochemical properties of NPP activity in cardiac synaptosomes and suggested the possible involvement of these enzymes in the control of nucleotides and nucleoside levels in the sympathetic nerve ends. In our study, NPP activities identified in soluble and microsomal fractions from heart provide information about a new important way to control the levels of adenine nucleotides, and consequently, its effects on cardiac functions.…”

“…The enzymatic action of NPP1-3 (in)directly results in the termination of nucleotide signalling, the salvage of nucleotides and/or the generation of new messengers like ADP, adenosine or pyrophosphate 17 . Recently, our group described the biochemical characteristics of this enzyme activity in synaptosomes from left ventricle of rat heart and identified the enzyme expression of the E-NPP family members in the cardiac tissue 20 . In that work, the co-expression of E-NPPs and E-NTPDases in cardiac synaptosomes was suggested as a multiple system for the control of extracellular nucleotide levels.…”