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A role for vessel wall ADP-ase in the maintenance of haemostasis has been suggested by a number of studies. For such a role to be likely the enzyme ideally should be an ecto-enzyme. The purpose of this study was to localise the ADP-ase using the technique of subcellular fractionation on a sucrose-density gradient in conjunction with marker enzyme assays for the different organelles.  Firstly, homogenates of cultured, pig aortic endothelial cells which had been harvested following the 5th-7th subculture were prepared. These were used to determine the kinetics of ADP-ase activity using the recently described rapid assay for ADP-ase which employs[β-s2P]ADP. The activity was found to be linear with respect to incubation time and protein concentration. The pH optimum was found to be 7.4 and the Km to be 45μM.  Post-nuclear supernatants derived from these homogenates (controls) and those from cells which had first been incubated with digitonin which binds to the membrane cholesterol thus making membranes heavier, were fractionated. In the controls, the profile of ADP-ase activity throughout the gradient was dissimilar to lactate dehydrogenase (cytosol), malate dehydrogenase (mitochondria), neutral α glucosidase (endoplasmic reticulum) and N-acetyl β glucosaminidase (lysosomes). It most closely corresponded to the 5’ nucleotidase profile (plasma membrane). Following digitonin treat ment, both 5’NT and ADP-ase shifted to a denser zone in the gradient confirming their coincident localisation.  Lastly, intact cells and sonicated cells were incubated with the enzyme inhibitor: diazotised sulphanalic acid which is a poorly permeant reagent. With intact cells, ADP-ase and 5’NT were inhibited whereas LDH was not, but with sonicated cells all three enzymes were inactivated.  Thus ADP-ase is an ecto-enzyme on the plasma membrane of pig aortic endothelial cells.

Subcellular Localisation Of ADP-ASE In Cultured, Porcine, Endothelial Cells