Human placental 5'-nucleotidase: purification and properties.

Subcellular fractionation of human term placenta showed that the highest relative specific activity of 5'-nucleotidase and alkaline phosphatase resided in the microsomal fraction; of the total 5'-nucleotidase activity present, 7 per cent was in the cytosol. 5'-Nucleotidase was reproducibly purified over 500-fold in 17 per cent yield from the insoluble component of homogenates of term placenta to give a single major glycoprotein with two minor inactive protein contaminants. Purified placental 5'-nucleotidase was free from non-specific or alkaline phosphatase, hydrolysed 12 to 22 mumol AMP/min/mg of protein at 30 degrees C, and was activated up to fivefold by Triton X-100. AMP, Km 5 to 7 microM, was the preferred substrate. The Arrhenius plot was biphasic, with activation energies of 21.7 and 49.7 kJ/mol above and below 36 degrees C, the region of the transition temperature. Nucleoside di- and triphosphates inhibited competitively; the most potent inhibitors were ADP and adenosine 5'-methylenediphosphonate, Ki slope 90 nm and 6 nm, respectively. Lectins inhibited the enzyme; concanavalin A caused time-dependent inactivation reversible by alpha-methyl-D-mannoside. EDTA inactivated the enzyme; partial reactivation was achieved with divalent cations. The pH optimum was 7.2 to 7.3; Mg2+ produced a second alkaline pH optimum. The properties of placental 5'-nucleotidase are those of an intrinsic membrane protein and, in general, resemble properties of the several 'ecto'-5'-nucleotidases which have been purified from other tissues, although certain differences in kinetic properties of the placental enzyme are apparent.