Summary
A role for vessel wall adenosine diphosphatase (ADPase) in limiting platelet aggregation
has been suggested by a number of studies. The purpose of this study was to determine
the properties and subcellular localization of ADPase in endothelial cells.
Homogenates were prepared from subcultured pig aortic endothelial cells and used to
determine the kinetics of ADPase activity with a recently described rapid assay employing
[²-32P]ADP. The activity was linear with incubation time and homogenate concentration.
The pH optimum was 7.4 and the apparent Km was 43 ¼M. The enzyme displayed an absolute
divalent cation requirement and its activity was unaffected by levamisole, a specific
alkaline phosphatase inhibitor, or by 2-glycerophosphate, a substrate for non-specific
phosphatases.
Post-nuclear supernatants were prepared from cell homogenates and were fractionated
by sucrose density centrifugation. The distribution of ADPase activity in the gradient
corresponded to 5'nucleotidase, a plasma membrane marker enzyme, and was clearly distinct
from the other organelle marker enzymes. When the cells were treated with the cholesterol-binding
agent, digitonin, before being homogenized, the activities of both 5'nucleotidase
and ADPase shifted to a denser zone in the gradient, confirming their coincident localization
to the plasma membrane.
Intact and sonicated cells were incubated with the poorly permeant enzyme inhibitor,
diazotized sulphanilic add. With intact cells, ADPase and 5'nucleotidase were inhibited
whereas lactate dehydrogenase, an intracellular enzyme, was unaffected; with sonicated
cells all three enzymes were inactivated. ADPase is thus an ecto-enzyme on the plasma
membrane of cultured pig aortic endothelial cells and is therefore ideally located
for a role in limiting platelet aggregation.
Key words
Adenosine diphosphate - Adenosine diphosphatase - Endothelial cells - Platelet aggregation
- Subcellular fractionation
