Phospholipase D (PLD) is a phosphodiesterase that hydrolyses membrane phospholipids generating phosphatidic acid (PA) and a polar head group. Activation of PLD represents a ubiquitous novel transmembrane signalling pathway in vascular endothelium. Recent studies in the P.I.'s laboratory as well as in many others, indicate that PLD activity is rapidly and markedly elevated upon receptor stimulation by a variety of agonists and by reactive oxygen species (ROS) or oxidants like hydrogen peroxide, fatty acid hydroperoxides and 4-hydroxynonenals. The mechanism(s) of PLD activation by oxidants involves probably protein tyrosine phosphorylation while agonist-mediated PLD activation is dependent on protein kinase C and Ca2+. The present proposal will focus on the characterization and purification of PLD from the lung tissue/endothelial cells to better understand the regulation of the enzyme during signal transduction.
The Specific Aims of the current proposal are: 1) to characterize PLD from bovine lung and bovine pulmonary artery endothelial cells (BPAEC); 2) to purify PLD from bovine lung and endothelial cell membrane; 3) to determine the role of protein phosphorylation in the regulation of PLD using reconstituted enzyme; 4) to identify and clone the PLD gene(s) from lung cDNA libraries. At present, PLD that is involved in signal transcription, has not been purified or cloned. Purification cloning and expression of the PLD will provide new and important tools to study the regulation and function of PLD activity and the gene(s) that encode it. Thus, the molecular and biochemical aspects of this proposal will further provide the necessary impetus for elucidating the role of PLD in cell signalling under normal conditions and vascular disorders.
