title: Biochemical characterization of an ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat cardiac soluble and microsomal fractions.
abstract: 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 K(M) 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 V(max) 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.
authors.affiliation: Laboratório de Estudos sobre o Sistema Purinérgico, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. danipochmann@hotmail.com
authors.forename: Daniela
authors.initials: D
authors.lastname: Pochmann
authors.forename: Adrine Maria
authors.initials: AM
authors.lastname: Innocente
authors.forename: Andréia
authors.initials: A
authors.lastname: Buffon
authors.forename: João José
authors.initials: JJ
authors.lastname: Freitas Sarkis
authors.forename: Lisiane De Oliveira
authors.initials: LO
authors.lastname: Porciúncula
mesh_terms: D000818:Animals; D006352:Heart Ventricles / Q000201:enzymology*; D006868:Hydrolysis; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D012995:Solubility; D013329:Structure-Activity Relationship; D013498:Suramin / Q000494:pharmacology
chemical_list: D013498:Suramin; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases