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.
D015720:5'-Nucleotidase; D002413:Cations, Divalent / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators / Q000494:pharmacology; D005260:Female; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D009708:Nucleotidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D017830:Octoxynol; D010920:Placenta / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011247:Pregnancy; D013379:Substrate Specificity
D002413:Cations, Divalent; D004793:Enzyme Reactivators; D037102:Lectins; D011092:Polyethylene Glycols; D017830:Octoxynol; D004492:Edetic Acid; D009708:Nucleotidases; C018775:adenosine monophosphatase; D015720:5'-Nucleotidase