Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects.
The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.
D000818:Animals; D004355:Drug Stability; D004546:Elapid Venoms / Q000378:metabolism*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000494:pharmacology; D013379:Substrate Specificity
D004546:Elapid Venoms; D004800:Enzymes, Immobilized; D008024:Ligands; D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2