The focus of this thesis is the control of phospholipase D (PLD) activation in permeabilized rat hepatocytes. PLD hydrolyzes phosphatidylcholine (PC) to form phosphatidic acid (PA) and free choline. In the presence of ethanol, the unusual phospholipid phosphatidylethanol (Peth) is produced, reaction which has been found in the tissues of animals exposed to ethanol, both acutely and chronically.
The first part of this thesis work was dedicated to investigating the optimal activation conditions for PLD--calcium and magnesium dependence as well as the role of protein cofactors, e.g. RhoA and ARF-1. There is a sharp increase in PLD activity in the permeabilized cells, in the presence of 10μM GTPγS, when the buffer contained 1mM free magnesium and 10nM free calcium. This PLD activity was further augmented by the addition of 1mM ATP. These data suggest that PLD isoforms with differing ion requirements might exist. In investigating the role of small G-proteins, we observed no PLD activity associated with ARF, but demonstrated a significant Rho dependent PLD activity.
The second phase of this thesis work was dedicated to investigating the ATP dependent activation of PLD. We observed PLD activity in the permeabilized cells when the cells were incubated with 1mM ATP in addition to 300mM EtOH. This unusual PLD activity is strictly calcium dependent. By using nonhydrolyzeable ATP analogs that are potent purinergic receptor agonists, we determined that the observed PLD activity was not a receptor mediated process.
To complete characterizing the ATP dependent PLD activity, we utilized suramin, reactive blue 2, PPADS and isoPPADS, specific purinergic receptor antagonists. In the intact cells, there was partial inhibition of the purinergic PLD, suggesting that a heterogeneous population of purinergic receptors reside on the membrane. In permeabilized cells, there was a complete inhibition of PLD (IC 50 Suramin=40μM). Using structural analogs of suramin, we demonstrated that PLD inhibition requires the entire suramin molecule, not just the charged portions.
Finally, we investigated the role of substrate presentation and phospholipid requirements for PLD utilization of exogenous substrate. In the permeabilized cells, PLD is able to hydrolyze exogenous PC presented in the form of a small unilamelar vesicle. When the vesicles contained 33%PE in addition to PC, there was an increase in PLD hydrolysis of the exogenous PC. When PIP 2 was added to the vesicles, we observed a loss of PLD activity. (Abstract shortened by UMI.)
