HUMAN PLACENTAL ATP-DIPHOSPHOHYDROLASE IS A HIGHLY N-GLYCOSYLATED PLASMA-MEMBRANE ENZYME

Human placental ATP diphosphohydrolase (ATP-DPH), has been previously characterized as an azide-sensitive, Ca2+- or Mg2+-dependent triphosph o- and diphosphonucleosidase which migrates as an 82 kDa protein band on SDS-PAGE (Christoforidis, S. et al. (1995) fur. J. Biochem. 234, 66 -74). In this paper we have studied the subcellular localization of pl acental ATP-DPH by differential centrifugation and flotation experimen ts. Using specific enzymatic markers it was found that ATP-DPH is loca lized on plasma membrane. ATP-DPH was found to be a highly N-glycosyla ted protein which is a common post-translational modification of plasm a membrane proteins. Extensive incubation of the native pure enzyme wi th N-glycosidase F resulted in the elimination of the 82 kDa form and the concurrent formation of a deglycosylated product of 57.5 kDa and f our other intermediate products, indicating the presence of at least f ive N-glycosylation sites within the ATP-DPH molecule. The partially d eglycosylated sample retained its activity in solution and in native g el electrophoresis and activity staining.