The relationship between high-affinity noncatalytic binding of snake venom phospholipases A2 to brain synaptic plasma membranes and their central lethal potencies.
The basic phospholipase A2 from Naja nigricollis (African spitting cobra) snake venom is enzymatically less active but more toxic than the acidic phospholipase A2 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 A2 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 Kd values of 1 X 10(-10) and 2.75 X 10(-8) M and Bmax values of 6 X 10(-13) and 3.4 X 10(-11) mol/mg, respectively. Multiple specific and nonspecific binding components were observed for each phospholipase A2; saturability for all of the binding sites was conclusively demonstrated only for the N. naja atra phospholipase A2 in a calcium-free medium (Bmax = 3.4 X 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 X 10(-8) M. These levels of binding (both total and specific) were directly correlated with the intraventricular lethal potencies of the phospholipases A2 (0.5 and 5.0 micrograms/rat for the N. nigricollis and N. naja atra phospholipases A2, 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 A2 or C suggested that radiolabeled phospholipase A2 binds to phospholipids rather than proteins. However, only the N. naja atra phospholipase A2 manifested a strict dependence on a divalent cation (Ca2+ or Sr2+) 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 A2, 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 A2.
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D004546:Elapid Venoms / Q000032:analysis* / Q000633:toxicity; D006358:Hot Temperature; D066298:In Vitro Techniques; D007276:Injections, Intraventricular; D007457:Iodine Radioisotopes; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D009186:Myelin Sheath / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013570:Synaptic Membranes / Q000378:metabolism*
D004546:Elapid Venoms; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium