Characterization of phospholipase D activation by GM2 activator in a cell-free system

Phospholipase D (PLD) activator which synergistically activates the enzyme with ADP ribosylation factor has recently been shown homologous to GM2 activator (Nakamura, S. et al.: Proc. Natl. Acad. Sci. USA 1998. 95, 12249/12253). The present studies were undertaken to further clarify the identity of the activator by immunological technique and to characterize the mechanism of activation of PLD by enzymological approach. The activator was further confirmed as GM2 activator by immunoblot analysis. Kinetic analysis showed Vmax for the PLD reaction was 16 fold elevated by GM2 activator, whereas Km for phosphatidylcholine remained constant by GM2 activator. These results strongly suggest that GM2 activator might activate enzyme by protein-protein interaction not by substrate modification. These results facilitate the understanding how the metabolism of both phospholipids and gangliosides is regulated by the same protein.