Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A 2 from Agkistrodon piscivorus piscivorus snake venom

Abstract

The basic Lys-49 phospholipase A 2 (PLA 2) from Agkistrodon pisciuorus piscivorus venom is homologous to the basic Asp-49 PLA 2 from the same venom as well as other snake venom PLA 2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent V max of the Lys-49 PLA 2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA 2, compared to the Asp-49 PLA 2 had < 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA 2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA 2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA 2. Evaluation of the present results, along with earlier findings using Asp-49 PLA 2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: (1) The A. p. piscivorus Asp-49 PLA 2 enzyme resembles the Asp-49 enzymes fron N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA 2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. (2) In contrast to some previous suggestions, basic PLA 2 enzymes are not necessarily more toxic than neutral or acidic enzymes. (3) Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA 2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA 2 enzymes exert effects independent of phospholipid hydrolysis.