Role of monomeric activators in cobra venom phospholipase A2 action.
Phospholipase A2 from cobra venom (Naja naja naja), which acts poorly on phosphatidylethanolamine (PE) in mixed micelles, is activated toward PE by the monomeric phospholipid dibutyrylphosphatidylcholine (dibutyryl-PC) which is an, even poorer substrate. Phosphorus-31 nuclear magnetic resonance spectroscopy was employed to show that only PE is hydrolyzed in mixtures of PE and dibutyryl-PC of various concentrations. The activation shows saturation behavior, and the fully activated enzyme hydrolyzes PE at a rate similar to its optimal substrate PC containing long chain fatty acid groups. Because dibutyryl-PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzymes by dibutyryl-PC rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Furthermore, if either PC pr PE as substrate is dispersed in mixed micelles, increasing amounts of the detergent Triton X-100 decrease the hydrolysis rate. The same detergent effect occurs if PE hydrolysis is activated by sphingomyelin (SPH). However, if the enzyme is activated by the monomeric dibutyryl-PC, this detergent effect can be overcome at high enough dibutyryl-PC concentrations. The hydrolysis of the monomeric dibutyryl-PC can also be stimulated by SPH in mixed micelles. This reaction shows no effect of detergent. Several models are considered to explain these observations, and it is suggested that the enzyme has two types of functional sites: an activator site and a catalytic site.
D004546:Elapid Venoms; D004789:Enzyme Activation; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D013109:Sphingomyelins / Q000494:pharmacology; D013997:Time Factors
D004546:Elapid Venoms; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013109:Sphingomyelins; D010767:Phosphorylcholine; C034826:1,2-dibutyrylphosphatidylcholine; D011092:Polyethylene Glycols; C028810:dodecylphosphocholine; D017830:Octoxynol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2