title: Properties of ecto-(inoganic) pyrophosphatase of nervous system cells in culture. Activation upon partial release of sialic acid from the cell surface.
abstract: Clonal line NN hamster astroblasts and clonal line N18 neuroblasts were treated with phospholipase C-free, protease-free, and hemolysin-free Clostridium perfringens sialidase, at a low level (5 X 10(-3) units/ml) so as to maintain cell intactness and to avoid spurious protein effects. A rapid, regular release of sialic acid was achieved. An approximately 9-fold increase in ecto-pyrophosphatase activity could be brought about by action of C. perfringens sialidase for 10 min. Since the sialidase preparations were employed at a level which gave a very low concentration of extraneous protein, and the preparations were free of demonstrable phospholipase C and protease activities, these effects appear to relate specifically to removal of cell surface sialic acid. Neutral p-nitrophenyl phosphatase was activated under the same conditions, but activity remained low compared with pyrophosphatase. Progress curves for activation of the two enzymes were dissimilar. Ecto-pyrophosphatase of NN and N18 cells had an absolute requirement for Mg2+ both before and after removal of cell surface sialic acid. In the presence of near optimum Mg2+ (5 mM), other divalent cations were inhibitory at a low level (10(-1)mM). The effect  of Mg2+ concentration, as well as inorganic pyrophosphate concentration, upon ecto-pyrophosphatase activity was shown to obey Michaelis-Menten kinetics for the control activity and for the sialidase-enhanced activity of both cell types. Km for Mg2+ and for pyrophosphate remained constant upon ecto-pyrophosphatase enhancement by sialic acid removal; increase in enzymatic activity was accounted for entirely by an increase in Vmax.
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009420:Nervous System / Q000187:drug effects / Q000201:enzymology*; D009439:Neuraminidase; D011755:Pyrophosphatases / Q000378:metabolism*; D012794:Sialic Acids / Q000502:physiology*
chemical_list: D012794:Sialic Acids; D009439:Neuraminidase; D011755:Pyrophosphatases; D008274:Magnesium