Kinetic characterization of an NTPDase and 5´-nucleotidase by a crude synaptosomal fraction isolated from rat heart.
Almeida, M. E. Almeida1; Rücker, B.1; Barreto Chaves, M. L. M.2 and Sarkis, J.J.F.1
1Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; 2Departamento de Anatomia, ICB, USP, São Paulo, SP, Brasil
The first suggestions that purines acted in a cardioprotective role came with the demonstration that adenosine mediated vasodilatation during hypoxia to increased blood flow and thus maintain oxygen delivery to the heart. Besides, adenosine is an important extracellular signalling molecule with aintithrombotic and antiinflamatory properties. In heart, ATP is involved in positive ionotropic effects, may induce various forms of arrhythmia besides hypertrophy and apoptosis. Traditionally, the inactivation of adenine nucleotides signaling is attributed to its breakdown by cell-membrane bound enzymes, classified as ATPases, ecto-apyrases and ecto-5’-nucleotidases. The objective of the present study is to characterize the enzymes involved in ATP, ADP e AMP hydrolysis in cardiac synaptosomes of adult male rats. The synaptosomal fraction was prepared as previously described by Aloyo et al, 1991. The reaction was initiated by the addition of nucleotides (1.0 mM ATP, ADP or AMP) after 10 minutes of pre-incubation at 37°C. The nucleotide hydrolysis was determined by the Malachite Green method. The protein concentration and the incubation time were chosen in order to ensure the linearity of the reaction. Then, about 6 mg/tube of protein and 6 minutes of incubation were used in enzymatic assays for ATP/ADP. The mitochondria ATPase inhibitors (olygomycin 2mg/mL and sodium azide 100 mM) were used in all enzymatic ATP assays. For the study of AMP hydrolysis, 10 mg/tube of protein and 10 min were used. Both enzymes were cation-dependent: NTPDase showed a preference for ion Ca2+, while 5´-nucleotidase showed a preference for ion Mg2+. In addition, both activities were blockade by metal ion chelator. KM and Vmax values for ATP, ADP and AMP hydrolysis were found to be 143 ± 16 mM and 564 ± 89 nmol Pi/min/mg protein (n=3), 68 ± 17 mM and 216 ± 37 nmol Pi/min/mg protein (n=4), 65 ± 9 mM and 68 ± 6 nmol nmol Pi/min/mg protein (n=3), respectively. There is evidence that purines contribute to a number of processes involved in normal cardiovascular function, and that disturbances in purinergic signaling are involved in some cardiovascular diseases. Then these results may represent a new insight about the purines participation on the cardiovascular system.
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