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

In this study, we have reported the kinetic and biochemical characterization of ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat cardiac fractions, one soluble and the other enriched in vesicles derived from sarcoplasmic reticulum. Both fractions demonstrated E-NPP activities, which could be observed by extracellular hydrolysis of p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) and other biochemical characteristics. The KM values for the hydrolysis of p-Nph-5′-TMP in soluble and microsomal fractions were 118.53 ± 27.28 and 91.92 ± 12.49 µM, respectively. The Vmax values calculated were 2.56 ± 0.15 and 113.87 ± 21.09 nmol p-nitrophenol/min/mg of protein in soluble and microsomal fractions, respectively. Among the compounds tested to evaluate the possible activity of other enzymes on p-Nph-5′-TMP hydrolysis, only suramin (0.25 mM) produced a significant inhibition of substrate hydrolysis. Thus, our results strongly suggest the presence of E-NPP enzymes in subcellular fractions of rat heart, which could be involved in nucleotide signalling in the cardiac tissue.