Abstract
5'-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein-Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5 mM AMP, 10 mM magnesium chloride, 7.2 mM lead nitrate, 0.1 M Tris-maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5'-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8 micron. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4 mM in pericentral zones and 0.8 mM in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.
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