We propose to characterize the interaction of three phospholipases (PLA) purified from liver mitochondrial PLA2, lysosomal PLA1, and plasma membrane PLA1 (hepatic lipase) with their substrates. Established procedures will be used for their purification. We will determine the specificity of these enzymes using several chemically- and biochemically-synthesized neutral lipids and phospholipids. These lipids will be studied as substrates in the monomeric as well as aggregate forms. The nature of the aggregate form will be studied using NMR spectroscopy. Likewise, we will study the charge characteristics of the substrates using microelectrophoresis. Kinetic analysis of their hydrolysis will be carried out using mixed micellar substrate suspensions. Considerable emphasis is placed on the structure of the mixed micelle and the enzyme-micelle binding. With these techniques, we anticipate that we will determine the following points: (1) substrate specificity, both acyl and polar head group; (2) substrate charge requirements for activity; (3) effect of physical structure (bilayer liposome, hexagonal array, micelles made with mixtures of Triton X100 and phospholipids) on activity; and (4) capacity of the enzymes to act on monomeric substrate. These studies will provide a detailed analysis of three distinct types of cellular phospholipases; these enzymes are involved in those membrane-related phenomena in which there is metabolism and turnover of associated phospholipids.
