The present study describes the enzymatic properties and molecular identification of 5′-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent Km (Michaelis constant) values calculated from the Eadie–Hofstee plot were 59.7±10.4 μM and 134.8±32.1 μM, with Vmax values of 6.7±0.4 and 143.8±23.8 nmol Pi/min/mg of protein (means±S.D., n=4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5′-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5′-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation–contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.

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