Interaction of Phospolipase A₂ form Naja melanoleuca Sanke Venom with Meoomeric Substrate Analogs

Abstract

Unlike porcine pancreatic phospholipase A,, the enzyme from Naja melanoleuca does not display biphasic kinetic behaviour at substrate concentrations around the critical inicelle concentration. This snake venom enzyme was further investigated by direct binding studies using n-tridecylphosphocholine. Binding of this substrate analog to the enzyme was monitored by using equilibrium gel filtration, equilibrium dialysis and ultraviolet difference spectroscopy. It is concluded that, in the presence of submicellar concentrations of n-tridecylphosphocholine, a lipid-protein complex is formed consisting of about 4 protein and 36 lipid molecules. CaZ + ions are required for the formation of this complex. A model is proposed which describes the formation of this type of complex. These lipidprotein aggregates are held responsible for the non-hyperbolic kinetic behaviour of the snake venom enzyme towards monomeric substrates.Phospholipase A, is widespread in Nature. The enzyme is abundant in pancreatic tissues as well as in the venoms of arthropods and snakes. Irrespective of the source, the enzymes show a high degree of homology. An identical mechanism for the hydrolysis of monomeric phospholipids has been postulated [I]. The activity of all phospholipases strongly depends on the physico-chemical state of the substrate. The enzyme displays Michaelis-Menten kinetics when acting on monomeric substrates. In the presence of certain organized lipidwater interfaces, phospholipase A, shows activities which can be three orders of magnitude higher than found for monomeric substrates. This results in a break in the velocity vs substrate concentration plots at the critical micelle concentration of the substrate [2].For the pancreatic enzymes it has been proposed by Verger et al. 131 that a particular surface region of the enzyme, called the interface recognition site (IRS), is responsible for the interaction with aggregated lipid-water interfaces.