The relationship between high-affinity noncatalytic binding of snake venom phospholipases A 2 to brain synaptic plasma membranes and their central lethal potencies

Abstract

The basic phospholipase A 2 from Naja nigricollis (African spitting cobra) snake venom is enzymatically less active but more toxic than the acidic phospholipase A 2 from Naja naja atra (Taiwan cobra) snake venom, following injection into the right lateral ventricle of the brain of rats. When radiolabeled with 125I, these phospholipases A 2 retained enzymatic activities and lethal potencies. Both enzymes bound with high affinity and specificity to brain synaptic plasma membrane preparations in vitro even in the absence of calcium, suggesting a non-catalytic binding. The acidic enzyme, in a calcium-free medium, had two binding components with K d values of 1.10·10 −10 and 2.75·10 −8 M and B max values of 6·10 −13 and 3.4·10 −11 mol/mg, respectively. Multiple specific and nonspecific binding components were observed for each phospholipase A 2; saturability for all of the binding sites was conclusively demonstrated only for the N. naja atra phospholipase A 2 in a calcium-free medium ( B max = 3.4·10 −11 mol/mg ). The levels of specific and total binding were 150 pmol/mg and 450 pmol/mg, respectively, for the comparatively toxic enzyme and 15 pmol/mg and 35 pmol/mg, respectively, for the comparatively nontoxic enzyme at a concentration of 2.5·10 −8 M. These levels of binding (both total and specific) were directly correlated with the intraventricular lethal potencies of the phospholipases A 2 (0.5 and 5.0 μg/rat for the N. nigricollis and N. naja atra phospholipases A 2, respectively), suggesting a possible relationship between binding and lethal potency. Carbamylation of lysines reduced the levels of binding and the lethal potencies of both enzymes to a greater extent than their enzymatic activities. Pretreatment with high temperature, proteinases, phospholipases A 2 or C suggested that radiolabeled phospholipase A 2 binds to phospholipids rather than proteins. However, only the N. naja atra phospholipase A 2 manifested a strict dependence on a divalent cation (Ca 2+ or Sr 2+) for most of its binding. The N. nigricollis enzyme demonstrated a much lower rate of dissociation from synaptic plasma membranes than did N. naja atra phospholipase A 2, suggesting that hydrophobic interactions are more important in the binding of the more toxic enzyme as compared to the less toxic enzyme. It is proposed that differences in the extent of high-affinity noncatalytic binding to membrane phospholipids may be at least partly responsible for the marked difference in central toxicities of these two phospholipases A 2.