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.