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 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.
Rücker
Bárbara
B
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul - UFRGS, Rua Ramiro Barcelos, 2600 - Prédio ANEXO, Porto Alegre, RS CEP 90035-003, Brazil.
Almeida
Manoela E
ME
Libermann
Towia A
TA
Zerbini
Luiz F
LF
Wink
Márcia R
MR
Sarkis
João José Freitas
JJ
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006352:Heart Ventricles / Q000201:enzymology*; D006868:Hydrolysis; D008297:Male; D009596:Nitrophenols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019810:Sodium Azide / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology; D013574:Synaptosomes / Q000201:enzymology*; D013938:Thymidine Monophosphate / Q000378:metabolism