COBRA VENOM CYTOTOXIN FREE OF PHOSPHOLIPASE-A, AND PTS EFFECT ON MODEL MEMBRANES AND T-LEUKEMIA-CELLS

Membrane-active toxins from snake venom have been used previously to s tudy protein-lipid interactions and to probe the physical and biochemi cal states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable p hospholipase A(2) (PLA(2)). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA(2) in buffers at pH values between 7 .0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA( 2) (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes was studie d by EPR of spin probes in oriented lipid multilayers and H-1-NMR of s onicated liposomes. The cytotoxin did not significantly affect the pac king of lipids in pure phosphatidylcholine (PC) membranes and in PC me mbranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced an increase in membrane permeability a nd formation of nonbilayer structures in PC membranes containing 40 mo l% of PA or CL. The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurka t cells and normal lymphocytes were killed equivalently when treated w ith 10(-9) M PLA(2) and 10(-5) M cytotoxin in combination. From its ef fect on model membranes and Jurkat cells, it is suggested that purifie d cytotoxin preferentially targets and disrupts membranes that are ric h in acidic phospholipids on the extracellular side of the plasma memb rane.