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Purification of two phospholipase A isoenzymes with anticoagulant activity from the venom of the cobra Naja naja siamensis

1980; Elsevier BV; Volume: 18; Issue: 4 Linguagem: Inglês

10.1016/0041-0101(80)90048-3

1879-3150

Evert Karlsson, Piamsook Pongsawasdi,

Marine Invertebrate Physiology and Ecology

Two non-lethal phospholipases A with anticoagulant activity were purified from the venom of Naja naja siamensis by ion-exchange chromatography on Bio-Rex 70, gel filtration on Sephadex G-75 and ion-exchange chromatography on sulphopropyl-Sephadex C-25. They have 119 amino acid residues, seven disulfides and a very similar composition. Their sequences might differ only at two positions. The phospholipases can exist in three forms, as a monomer and two different aggregates, which behave differently in ion-exchange chromatography and short time analytical electrofocusing. A single phospholipase can therefore appear to be inhomogeneous. Upon preparative electrofocusing run for a long time each enzyme focused into a single zone with isolelectric points of 4·76 and 5·41, respectively. Basic phospholipases A are in general much more lethal than neutral or acidic ones. Some basic phospholipases are myotoxins and presynaptic neurotoxins, and it is proposed that the basic regions of the molecules facilitate the binding of these toxins to the negatively charged muscle and nerve membranes. A similar correlation does not exist with respect to the anticoagulant activity, since it was shown that a basic phospholipase A can be a powerful anticoagulant while another equally basic enzyme is a very poor one. Modification with p-bromophenacyl bromide suppresses both the phospholipase A and anti-coagulant activity. That does not necessarily mean that the anticoagulant activity is a consequence of the enzymic action, i.e. caused by the hydrolysis products or by destruction of essential phospholipids. Loss of anticoagulant activity may rather indicate that the modified protein is no longer capable of binding phospholipids. According to present hypothesis, phospholipases A inhibit coagulation by binding to phospholipids, which normally complex with many coagulation factors and thereby accelerate their activation.